The upstream region of the FOX3 gene encoding peroxisomal 3-oxoacyl-coenzyme A thiolase in Saccharomyces cerevisiae contains ABF1- and replication protein A-binding sites that participate in its regulation by glucose repression.

Expression of the FOX3 gene, which encodes yeast peroxisomal 3-oxoacyl-coenzyme A thiolase, can be induced by oleate and repressed by glucose. Previously, we have shown that induction was mediated by an oleate response element. Just upstream of this element a negatively acting control region that mediated glucose repression was found. In order to study this negative control region, we carried out DNA-binding assays and analyzed phenotypes of mutations in this region and in the trans-acting factor CAR80, which is identical to UME6. DNA-binding assays showed that two multifunctional yeast proteins, ABF1 and RP-A, interacted with the negative control element independently of the transcriptional activity of the FOX3 gene. ABF1 and RP-A, the latter being identical to BUF, were able to bind to DNA independently of one another but also simultaneously. The phenotypes of mutations in either DNA-binding sites of ABF1, RP-A, or both, which affected the DNA binding of these factors in vitro, indicated that these sites and the proteins that interact with them participate in glucose repression. The involvement of the RP-A site in glucose repression was further supported by our observation that the CAR80 gene product, which is required for repression mediated by the RP-A site, was essential for maintenance of glucose repression. In addition to the RP-A site in the FOX3 promoter, similar sequences were observed in other genes involved in peroxisomal function. RP-A proved to bind to all of these sequences, albeit with various affinities. From these results it is concluded that the ABF1 and RP-A sites are being required in concert to mediate glucose repression of the FOX3 gene. In addition, coordinated regulation of expression of genes involved in peroxisomal function in response to glucose is mediated by proteins associated with the RP-A site, probably RP-A and CAR80.

Dynamics of gene expression revealed by comparison of serial analysis of gene expression transcript profiles from yeast grown on two different carbon sources.

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 (>20%) 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 approximately 6300 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 beta-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.

A collaborative EDNAP exercise on SNaPshot™-based mtDNA control region typing.

A collaborative European DNA Profiling (EDNAP) Group exercise was undertaken to assess the performance of an earlier described SNaPshot™-based screening assay (denoted mini-mtSNaPshot) (Weiler et al., 2016) [1] that targets 18 single nucleotide polymorphism (SNP) positions in the mitochondrial (mt) DNA control region and allows for discrimination of major European mtDNA haplogroups. Besides the organising laboratory, 14 forensic genetics laboratories were involved in the analysis of 13 samples, which were centrally prepared and thoroughly tested prior to shipment. The samples had a variable complexity and comprised straightforward single-source samples, samples with dropout or altered peak sizing, a point heteroplasmy and two-component mixtures resulting in one to five bi-allelic calls. The overall success rate in obtaining useful results was high (97.6%) given that some of the participating laboratories had no previous experience with the typing technology and/or mtDNA analysis. The majority of the participants proceeded to haplotype inference to assess the feasibility of assigning a haplogroup and checking phylogenetic consistency when only 18 SNPs are typed. To mimic casework procedures, the participants compared the SNP typing data of all 13 samples to a set of eight mtDNA reference profiles that were described according to standard nomenclature (Parson et al., 2014) [2], and indicated whether these references matched each sample or not. Incorrect scorings were obtained for 2% of the comparisons and derived from a subset of the participants, indicating a need for training and guidelines regarding mini-mtSNaPshot data interpretation.

Expression of genes encoding peroxisomal proteins in Saccharomyces cerevisiae is regulated by different circuits of transcriptional control.

In Saccharomyces cerevisiae induction of the FOX3 gene, encoding peroxisomal 3-oxoacyl-CoA thiolase, by growth on oleate as sole carbon source, is exerted via the cis-acting DNA element designated oleate response element (ORE) (Einerhand et al. (1991) Eur. J. Biochem. 200, 113-122). The transcription factor(s) binding to this upstream activation site (UAS) are still unknown, however. Induction of another peroxisomal enzyme, citrate synthase (CIT2) is dependent on the products of two genes called RTG1 and RTG2 (Liao and Butow (1993) Cell 72, 61-71). In the present study we have investigated whether RTG1 controls other genes coding for peroxisomal proteins, and whether such control takes place via the ORE. A number of genes coding for a variety of peroxisomal proteins such as: thiolase and catalase (peroxisomal matrix proteins), PAS3p (a peroxisomal membrane protein) and PAS10p (a protein involved in the import of peroxisomal proteins) were studied in their response to RTG1. Although the RTG1 and 2 products proved to be required for the increase in number and volume of peroxisomes upon induction by oleate, the single promoter output of the chosen set of genes remained practically unchanged in a rtg1 mutant strain. In addition gel retardation experiments indicated that RTG1 does not bind to the ORE. The behavior of genes coding for the various proteins also varied during repression, derepression and induction, indicating that probably a number of proteins are involved in tuning the output of each gene to cellular demand.

In silicio search for genes encoding peroxisomal proteins in Saccharomyces cerevisiae.

The biogenesis of peroxisomes involves the synthesis of new proteins that after, completion of translation, are targeted to the organelle by virtue of peroxisomal targeting signals (PTS). Two types of PTSs have been well characterized for import of matrix proteins (PTS1 and PTS2). Induction of the genes encoding these matrix proteins takes place in oleate-containing medium and is mediated via an oleate response element (ORE) present in the region preceding these genes. The authors have searched the yeast genome for OREs preceding open reading frames (ORFs), and for ORFs that contain either a PTS1 or PTS2. Of the ORFs containing an ORE, as well as either a PTS1 or a PTS2, many were known to encode bona fide peroxisomal matrix proteins. In addition, candidate genes were identified as encoding putative new peroxisomal proteins. For one case, subcellular location studies validated the in silicio prediction. This gene encodes a new peroxisomal thioesterase.

The Drosophila brahma complex is an essential coactivator for the trithorax group protein zeste.

The trithorax group (trxG) of activators and Polycomb group (PcG) of repressors are believed to control the expression of several key developmental regulators by changing the structure of chromatin. Here, we have sought to dissect the requirements for transcriptional activation by the Drosophila trxG protein Zeste, a DNA-binding activator of homeotic genes. Reconstituted transcription reactions established that the Brahma (BRM) chromatin-remodeling complex is essential for Zeste-directed activation on nucleosomal templates. Because it is not required for Zeste to bind to chromatin, the BRM complex appears to act after promoter binding by the activator. Purification of the Drosophila BRM complex revealed a number of novel subunits. We found that Zeste tethers the BRM complex via direct binding to specific subunits, including trxG proteins Moira (MOR) and OSA. The leucine zipper of Zeste mediates binding to MOR. Interestingly, although the Imitation Switch (ISWI) remodelers are potent nucleosome spacing factors, they are dispensable for transcriptional activation by Zeste. Thus, there is a distinction between general chromatin restructuring and transcriptional coactivation by remodelers. These results establish that different chromatin remodeling factors display distinct functional properties and provide novel insights into the mechanism of their targeting.

A heterodimer of the Zn2Cys6 transcription factors Pip2p and Oaf1p controls induction of genes encoding peroxisomal proteins in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, two transcriptional activators belonging to the Zn2Cys6 protein family, Pip2p and Oaf1p, 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 x Oaf1p heterodimer, but in cells in which Oaf1p was overexpressed an Oaf1p homodimer was also observed. The genes encoding Oaf1p and Pip2p were controlled in different ways. The OAF1 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 x Oaf1p heterodimer is responsible for the strong expression of the genes encoding peroxisomal proteins upon growth on oleate. Pip2p and Oaf1p form an example of a heterodimere of yeast Zn2Cys6 zinc-finger proteins binding to DNA.

Mannose 6-phosphate-independent targeting of cathepsin D to lysosomes in HepG2 cells.

We have studied the role of N-linked oligosaccharides and proteolytic processing on the targeting of cathepsin D to the lysosomes in the human hepatoma cell line HepG2. In the presence of tunicamycin cathepsin D was synthesized as an unglycosylated 43-kDa proenzyme which was proteolytically processed via a 39-kDa intermediate to a 28-kDa mature form. Only a small portion was secreted into the culture medium. During intracellular transport the 43-kDa procathepsin D transiently became membrane-associated independently of binding to the mannose 6-phosphate receptor. Subcellular fractionation showed that unglycosylated cathepsin D was efficiently targeted to the lysosomes via intermediate compartments similar to the enzyme in control cells. The results show that in HepG2 cells processing and transport of cathepsin D to the lysosomes is independent of mannose 6-phosphate residues. Inhibition of the proteolytic processing of 53-kDa procathepsin D by protease inhibitors caused this form to accumulate intracellularly. Subcellular fractionation revealed that the procathepsin D was transported to lysosomes, thereby losing its membrane association. Procathepsin D taken up by the mannose 6-phosphate receptor also transiently became membrane-associated, probably in the same compartment. We conclude that the mannose 6-phosphate-independent membrane-association is a transient and compartment-specific event in the transport of procathepsin D.

The DNA binding domain (POU domain) of transcription factor oct-1 suffices for stimulation of DNA replication.

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.

A homeotic mutation in the trithorax SET domain impedes histone binding.

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 oct-1 homeo domain contacts only part of the octamer sequence and full oct-1 DNA-binding activity requires the POU-specific domain.

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 Kd 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.

Pip2p: a transcriptional regulator of peroxisome proliferation in the yeast Saccharomyces cerevisiae.

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.

Peroxisomal beta-oxidation of polyunsaturated fatty acids in Saccharomyces cerevisiae: isocitrate dehydrogenase provides NADPH for reduction of double bonds at even positions.

The beta-oxidation of saturated fatty acids in Saccharomyces cerevisiae is confined exclusively to the peroxisomal compartment of the cell. Processing of mono- and polyunsaturated fatty acids with the double bond at an even position requires, in addition to the basic beta-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 beta-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 NADP-dependent 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 Deltaidp3 cells.