Disease relevance of ROX1

• To determine whether the Rox1 repressor bound DNA, the gene was expressed in Escherichia coli cells as a fusion to the maltose-binding protein and this fusion was partially purified by amylose affinity chromatography [1].
• Only three ORFs are significantly repressed by Rox1 but they cannot be considered as typical hypoxic genes because they are not overexpressed during hypoxia [2].

High impact information on ROX1

• DNA-binding repressor proteins mediate regulation of yeast genes by cell type (Mcm1/alpha 2 and a1/alpha 2), glucose (Mig1) and oxygen (Rox1) (refs 1-4 respectively) [3].
• Expression of GPD2 is also unaffected by ROX1 and ROX3, encoding putative regulators of hypoxic and stress-controlled gene expression [4].
• A number of trans-acting mutations affecting a gene designated ROX1 caused constitutive expression of both the fused and wild-type genes, indicating that the ROX1 gene product operates through the ANB1 modulator sequence at the level of transcription [5].
• Rfg1, a protein related to the Saccharomyces cerevisiae hypoxic regulator Rox1, controls filamentous growth and virulence in Candida albicans [6].
• Repression of two other hypoxic genes in which Mot3 sites were associated with Rox1 sites was reduced in the deletion strain, but other hypoxic genes were unaffected [7].

Biological context of ROX1

• Finally, ROX1 was mapped to yeast chromosome XVI, near the ARO7-OSM2 locus [1].
• Oxygen levels are sensed by changes in heme biosynthesis, which controls the transcription of the ROX1 gene, encoding a protein that binds to the regulatory region of each hypoxic gene to repress transcription [8].
• Under aerobic conditions oxygen control of gene expression is exerted through the activator Hap1 and the repressor Rox1 [9].
• One is Rox1, an HMG protein, and the second, originally designated Rox7, is shown here to be Mot3, a global C2H2 zinc finger regulator [10].
• The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROX1 factor is mediated by an upstream repression sequence consisting of a T-rich segment adjacent to the consensus elements that are present in the 5' flanking regions of several other yeast genes [11].

Associations of ROX1 with chemical compounds

• Evidence that ROX1 binds to DNA or is part of a DNA-binding complex is described [12].
• Repression of ERG11 expression was dependent upon the ROX1 repressor and additional repressor(s) designated as Old (overexpression of lanosterol demethylase) [13].
• We report that ROX1 deletion resulted in 2.5- to 16-fold-lower susceptibilities to azoles and terbinafine [14].
• Rox1 mediated repression. Oxygen dependent repression in yeast [15].
• We report here cross-regulation between Rox1 and Mot3 and Rfx1 in the regulation of the RNR genes encoding ribonucleotide diphosphate reductase [16].

Physical interactions of ROX1

• This hypothesis was confirmed by using a set of fusions between sequences encoding the GAL4 DNA-binding domain and portions of ROX1 [17].
• To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene [18].
• Under hypoxic conditions, heme levels fall, and a heme-deficient Hap1 complex represses ROX1 expression [19].
• Chromatin immunoprecipitation assays indicated that Rox1 stabilized Mot3 binding to DNA [20].
• The hypoxic genes of Saccharomyces cerevisiae are transcriptionally repressed during aerobic growth through recruitment of the Ssn6/Tup1 general repression complex by the DNA binding protein Rox1 [20].

Regulatory relationships of ROX1

• In the absence of heme, Cyp1p activated HEM13 and strongly repressed ROX1, allowing de-repression of HEM13 [21].
• In Saccharomyces cerevisiae the anaerobic (oxygen-repressed) ANB1 gene and a group of aerobic (oxygen-induced) genes are coordinately regulated by the ROX1 gene [22].
• Finally, FET4 expression is regulated in response to oxygen by the Rox1 repressor [23].
• The ROX1 locus of S. cerevisiae is known to regulate CYC1, COXVb, and ANB1 genes at the transcriptional level; the ROX1 locus thus regulates all known anaerobically expressed genes that are involved in different cellular functions such as respiration and protein synthesis [24].
• The heme-Hap1 complex activates transcription of the ROX1 gene that encodes a repressor of one set of hypoxic genes [19].

Other interactions of ROX1

• The regulatory region of one well-studied hypoxic gene, ANB1, is comprised of two operators, OpA and OpB, each of which has two strong Rox1 binding sites, yet OpA represses transcription almost 10 times more effectively than OpB [7].
• With regard to the effect brought about by the deletion of rox1 and srb10, correspondence cluster analysis revealed that the transcriptome profile in aerobic conditions is very similar in the wild-type and both deletion strains [25].
• The repression of hypoxic genes under normoxic conditions results from Hap1-mediated activation of ROX1 transcription [9].
• Repression of anaerobic genes results from co-induction of Mot3 and Rox1 in aerobic cells [10].
• Furthermore, oxygen repression of the hypoxic HEM13 gene was not affected by the deletion nor was this putative ROX1 gene regulated positively by oxygen as is the case for the S. cerevisiae gene [26].

Analytical, diagnostic and therapeutic context of ROX1

• By using a gel retardation assay, both the fusion protein and Rox1 itself were found to bind specifically to a synthetic 32-bp DNA containing the hypoxic consensus sequence [1].
• Sequence analyses of ROX1 in reo1 strains reveal a frame-shift mutation in the 5'-end of its coding region, resulting in a nonsense codon in the sixth position [27].
• DNA arrays were used to investigate the functional role of Rox1 in mediating acclimatization to anaerobic conditions in Saccharomyces cerevisiae [28].
• Northern-blot analyses of a reo1 strain show wild-type expression of the aerobic genes examined, but de-repression of the Rox1-regulated, hypoxic genes [27].
• To better understand the structure of Rox1 and how it interacts with DNA, 38 missense mutations in the HMG domain were isolated through a combination of random and site-directed mutageneses, the latter directed to two Ile residues that play an important role in DNA recognition and bending by HMG domains [29].

References