We describe a genome-wide characterization of mRNA transcript levels in yeast grown on the fatty acid oleate, determined using Serial Analysis of Gene Expression (SAGE). Comparison of this SAGE library with that reported for glucose grown cells revealed the dramatic adaptive response of yeast to a change in carbon source. A major fraction (Ͼ20%) of the 15,000 mRNA molecules in a yeast cell comprised differentially expressed transcripts, which were derived from only 2% of the total number of ϳ6300 yeast genes. Most of the mRNAs that were differentially expressed code for enzymes or for other proteins participating in metabolism (e.g., metabolite transporters). In oleate-grown cells, this was exemplified by the huge increase of mRNAs encoding the peroxisomal ␤-oxidation enzymes required for degradation of fatty acids. The data provide evidence for the existence of redox shuttles across organellar membranes that involve peroxisomal, cytoplasmic, and mitochondrial enzymes. We also analyzed the mRNA profile of a mutant strain with deletions of the PIP2 and OAF1 genes, encoding transcription factors required for induction of genes encoding peroxisomal proteins. Induction of genes under the immediate control of these factors was abolished; other genes were up-regulated, indicating an adaptive response to the changed metabolism imposed by the genetic impairment. We describe a statistical method for analysis of data obtained by SAGE.

No abstract

In Saccharomyces cerevisiae, peroxisomes are the exclusive site for the degradation of fatty acids. Upon growth with the fatty acid oleic acid as sole carbon source, not only are the enzymes of beta‐oxidation and catalase A induced, but also the peroxisomal compartment as a whole increases in volume and the number of organelles per cell rises. We previously identified a cis‐acting DNA sequence [oleate response element (ORE)] involved in induction of genes encoding peroxisomal proteins. The aim of our investigation was to test whether a single mechanism acting via the ORE coordinates the events necessary for the proliferation of an entire organelle. Here we report the cloning and characterization of the oleate‐specific transcriptional activator protein Pip2p (pip: peroxisome induction pathway). Pip2p contains a typical Zn(2)‐Cys(6) cluster domain and binds to OREs. A pip2 deletion strain is impaired in growth on oleate as sole carbon source and the induction of beta‐oxidation enzymes is abolished. Moreover, only a few, small peroxisomes per cell can be detected. These results indicate that fatty acids activate Pip2p, which in turn activates the transcription of genes encoding beta‐oxidation components and acts as the crucial activator of peroxisomes.

In the yeast Saccharornyces cerevisiae, two transcriptional activators belonging to the Zn,Cys, protein family, Pip2p and Oaflp, are involved in fatty-acid-dependent induction of genes encoding peroxisomal proteins. This induction is mediated via an upstream activation sequence called the oleate-response element (ORE). DNA-bandshift experiments with ORE probes and epitope-tagged proteins showed that two binary complexes occurred: in wild-type cells the major complex consisted of a Pip2p . Oaflp heterodimer, but in cells in which Oaflp was overexpressed an Oaflp homodimer was also observed. The genes encoding Oaflp and Pip2p were controlled in different ways. The OAFl gene was constitutively expressed, while the PIP2 gene was induced upon growth on oleate, giving rise to positive autoregulatory control. We have shown that the Pip2p . Oaflp heterodimer is responsible for the strong expression of the genes encoding peroxisomal proteins upon growth on oleate. Pip2p and Oaflp form an example of a heterodimere of yeast Zn,Cys, zinc-finger proteins binding to DNA.

The β-oxidation of saturated fatty acids in Saccharomyces cerevisiae is confined exclusively to the peroxisomal compartment of the cell. Processing of monoand polyunsaturated fatty acids with the double bond at an even position requires, in addition to the basic β-oxidation machinery, the contribution of the NADPH-dependent enzyme 2,4-dienoyl-CoA reductase. Here we show by biochemical cell fractionation studies that this enzyme is a typical constituent of peroxisomes. As a consequence, the β-oxidation of mono-and polyunsaturated fatty acids with double bonds at even positions requires stoichiometric amounts of intraperoxisomal NADPH. We suggest that NADP-dependent isocitrate dehydrogenase isoenzymes function in an NADP redox shuttle across the peroxisomal membrane to keep intraperoxisomal NADP reduced. This is based on the finding of a third NADPdependent isocitrate dehydrogenase isoenzyme, Idp3p, next to the already known mitochondrial and cytosolic isoenzymes, which turned out to be present in the peroxisomal matrix. Our proposal is strongly supported by the observation that peroxisomal Idp3p is essential for growth on the unsaturated fatty acids arachidonic, linoleic and petroselinic acid, which require 2,4-dienoyl-CoA reductase activity. On the other hand, growth on oleate which does not require 2,4-dienoyl-CoA reductase, and NADPH is completely normal in Δidp3 cells.

The ubiquitous octamer-binding protein oct-1 contains a POU domain required for DNA binding, which can be subdivided into a POU-specific domain and a POU homeo domain. We have overproduced the POU domain and the POU homeo domain in a vaccinia expression system, purified both polypeptides to near homogeneity, and compared their DNA-binding properties. In contrast to the POU domain, the homeo domain protects only part of the octamer sequence in the Ad2 origin against breakdown by DNase I or hydroxyl radicals. Analysis of purine contacts by DMS and DEPC interference assays shows that the Ad2 octamer can be divided into two regions: one that is recognized both by the POU domain and the homeo domain in an identical fashion, and one that is only recognized by the POU domain. This suggests that the POU-specific domain is responsible for the additional contacts located at one side of the octamer. In agreement with this, mutating the first 3 nucleotides (ATG) of the octamer affected binding by the POU domain but not by the homeo domain. The apparent binding affinities to different octamer sites were compared. The homeo domain binds 600-fold less efficiently to the canonical octamer sequence (ATGCAAAT) than the POU domain. The difference is only sevenfold for the Ad2 octamer, whereas both K^ values are almost identical for the HSV ICP4 TAATGARAT motif. Both the POU and homeo domains recognize target sequences for mammalian homeo box proteins. We conclude that the octamer can act as a bipartite recognition sequence for oct-1 and that the POU-specific domain contributes to the binding affinity, as well as to the specificity, by providing additional contacts.

Oct‐1, also referred to as NFIII, OTF‐1, OBP100 or NF‐A1, is a ubiquitous sequence‐specific DNA binding protein that activates transcription and adenovirus DNA replication. The protein contains a conserved DNA binding domain (POU domain) present in several transcription factors. We have overproduced oct‐1, the related oct‐2 and several oct‐1 deletion mutants in a vaccinia expression system to identify the domains important for activation of DNA replication in vitro. Both oct‐1 and oct‐2 stimulate adenovirus DNA replication in an octamer‐dependent manner. From deletion studies it appears that the 160 amino acid long POU domain suffices for stimulation. This domain consists of two subdomains, a POU‐specific and a homeo domain. Deletion of the POU‐specific domain revealed that the homeo domain has an intrinsic, but weak DNA binding activity and surprisingly, inhibits DNA replication. As the POU domain does not coincide with the transcription activation domain, these results indicate that, although oct‐1 functions both in DNA replication and transcription, the mechanisms underlying these processes are probably distinct.

Trithorax (TRX) is a Drosophila SET domain protein that is required for the correct expression of homeotic genes. Here, we show that the TRX SET domain efficiently binds to core histones and nucleosomes. The primary target for the SET domain is histone H3 and binding requires the N-terminal histone tails. The previously described trx Z11 mutation changes a strictly conserved glycine in the SET domain to serine and causes homeotic transformations in the fly. We found that this mutation selectively interferes with histone binding, suggesting that histones represent a critical target during developmental gene regulation by TRX. The Polycomb group (PcG) of repressors and trithorax group (trxG) of activators target chromatin in order to "freeze" a mitotically stable pattern of gene expression and determined cell fate (Pirrotta 1998;Lyko and Paro 1999;Mahmoudi and Verrijzer 2001). The founding member of the trxG, the Drosophila trx gene, is required throughout development and controls the expression of several developmental regulators, including the homeotic genes (Ingham and Whittle 1980;Ingham 1985;Breen 1999). trx is related to the human Mixed Lineage Leukemia (MLL) gene, which is involved in translocations associated with the majority of cases of infant leukemias (Waring and Cleary 1997). TRX and MLL are part of a highly conserved regulatory network that is required for the correct expression of the homeotic selector genes and determination of segment identity in both mammals and Drosophila. They are very large proteins that contain structural motifs common to chromatin-associated factors such as PHD fingers and a C-terminal SET domain ( Fig. 1A; Mazo et al. 1990;Stassen et al. 1995).The SET domain is a highly conserved 130-150 amino acids motif initially recognized as a common element in chromatin regulators with opposing activities: the suppressor of position affect variegation Su(var)3-9, the PcG protein Enhancer of Zeste [E(z)], and TRX (Jenuwein et al. 1998). The SET domain has been implicated in a multitude of different protein-protein interactions and functions. The SET domains of MLL, yeast Set1p, and E(z) bind to myotubularin-related dual-specificity phosphatases and anti-phosphatases that modulate growth control (Cui et al. 1998). The TRX and MLL SET domains bind to the SNF5 component of the ATP-dependent remodeler SWI/SNF (Rozenblatt-Rosen et al. 1998) and mediate self-association (Rozovskaia et al. 2000). Furthermore, the SET domain of yeast Set1p binds the Mec3p checkpoint protein and has been implicated in DNA repair and telomere function (Corda et al. 1999). The Set1p SET domain alone suffices to mediate telomeric silencing, suggesting that it forms a functional unit (Nislow et al. 1997). Recently, it was shown that SUV39H1, the mammalian homolog of Su(var)3-9, selectively methylates lysine 9 of histone H3 (Rea et al. 2000;Jenuwein 2001). This modification creates a binding site for HP1 and thus can contribute to the propagation of a heterochromatin domain (Bannister et al. 2001;Lachner et al. 2001;...