Abstract
A few yeasts, including Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators (YNA1, YNA2). Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems.
Highlights
IntroductionThe genes involved in nitrate assimilation are inducible by the substrate and encode nitrate reductase (YNR1), nitrite reductase (YNI1) and a high affinity nitrate transporter (YNT1)
The least repressive effect is exerted by proline as the steady state mRNA levels on proline plus nitrate are from 40% to 85% of those obtained upon incubation on nitrate alone
We have confirmed, refined and extended these findings, establishing that the appearance of the YNT1, YNI1 and YNR1 mRNAs follows that of the transcriptional activator genes YNA1 and YNA2
Summary
The genes involved in nitrate assimilation are inducible by the substrate and encode nitrate reductase (YNR1), nitrite reductase (YNI1) and a high affinity nitrate transporter (YNT1). These co-regulated genes reside in a cluster together with the two pathway-specific regulatory genes YNA1 and YNA2 [2] (Fig 1A). As in A. nidulans and N. crassa, in H. polymorpha the expression of the transporter and enzymes involved in nitrate assimilation is subject to both nitrate induction and nitrogen metabolite repression, which blocks the nitrate pathway when the products of the reduction process such as ammonium or glutamine, are available in the growth medium [2,5,6,7,8,9,10,11,12,13]. Martìn and co-workers (2011) identified and described the NPR1 gene, encoding the Npr (Nitrogen Permease Reactivator 1) kinase involved in Ynt phosphorylation and regulation in response to the nitrogen and carbon source availability [14,15]
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