Rosa Passantino

Hypoxia Response Elements in the Aldolase A, Enolase 1, and Lactate Dehydrogenase A Gene Promoters Contain Essential Binding Sites for Hypoxia-inducible Factor 1

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor which is expressed when mammalian cells are subjected to hypoxia and which activates transcription of genes encoding erythropoietin, vascular endothelial growth factor, and other proteins that are important for maintaining oxygen homeostasis. Previous studies have provided indirect evidence that HIF-1 also regulates transcription of genes encoding glycolytic enzymes. In this paper we characterize hypoxia response elements in the promoters of the ALDA, ENO1, and Ldha genes. We demonstrate that HIF-1 plays an essential role in activating transcription via these elements and show that although absolutely necessary, the presence of a HIF-1 binding site alone is not sufficient to mediate transcriptional responses to hypoxia. Analysis of hypoxia response elements in the ENO1 and Ldha gene promoters revealed that each contains two functionally-essential HIF-1 sites arranged as direct and inverted repeats, respectively. Our data establish that functional hypoxia-response elements consist of a pair of contiguous transcription factor binding sites at least one of which contains the core sequence 5′-RCGTG-3′ and is recognized by HIF-1. These results provide further evidence that the coordinate transcriptional activation of genes encoding glycolytic enzymes which occurs in hypoxic cells is mediated by HIF-1.

ENO1 gene product binds to the c-myc promoter and acts as a transcriptional repressor: relationship with Myc promoter-binding protein 1 (MBP-1)

The Myc promoter‐binding protein‐1 (MBP‐1) is a 37–38 kDa protein that binds to the c‐myc P2 promoter and negatively regulates transcription of the protooncogene. MBP‐1 cDNA shares 97% similarity with the cDNA encoding the glycolytic enzyme α‐enolase and both genes have been mapped to the same region of human chromosome 1, suggesting the hypothesis that the two proteins might be encoded by the same gene. We show here data indicating that a 37 kDa protein is alternatively translated from the full‐length α‐enolase mRNA. This shorter form of α‐enolase is able to bind the MBP‐1 consensus sequence and to downregulate expression of a luciferase reporter gene under the control of the c‐myc P2 promoter. Furthermore, using α‐enolase/green fluorescent protein chimeras in transfection experiments we show that, while the 48 kDa α‐enolase mainly has a cytoplasmic localization, the 37 kDa α‐enolase is preferentially localized in the cell nuclei. The finding that a transcriptional repressor of the c‐myc oncogene is an alternatively translated product of the ENO1 gene, which maps to a region of human chromosome 1 frequently deleted in human cancers, makes ENO1 a potential candidate for tumor suppressor.

NEGATIVE REGULATION OF BETA ENOLASE GENE TRANSCRIPTION IN EMBRYONIC MUSCLE IS DEPENDENT UPON A ZINC FINGER FACTOR THAT BINDS TO THE G-RICH BOX WITHIN THE MUSCLE-SPECIFIC ENHANCER

We have previously identified a muscle-specific enhancer within the first intron of the human β enolase gene. Present in this enhancer are an A/T-rich box that binds MEF-2 protein(s) and a G-rich box (AGTGGGGGAGGGGGCTGCG) that interacts with ubiquitously expressed factors. Both elements are required for tissue-specific expression of the gene in skeletal muscle cells. Here, we report the identification and characterization of a Kruppel-like zinc finger protein, termed β enolase repressor factor 1, that binds in a sequence-specific manner to the G-rich box and functions as a repressor of the β enolase gene transcription in transient transfection assays. Using fusion polypeptides of β enolase repressor factor 1 and the yeast GAL4 DNA-binding domain, we have identified an amino-terminal region responsible for the transcriptional repression activity, whereas a carboxyl-terminal region was shown to contain a potential transcriptional activation domain. The expression of this protein decreases in developing skeletal muscles, correlating with lack of binding activity in nuclear extract from adult skeletal tissue, in which novel binding activities have been detected. These results suggest that in addition to the identified factor, which functionally acts as a negative regulator and is enriched in embryonic muscle, the G-rich box binds other factors, presumably exerting a positive control on transcription. The interplay between factors that repress or activate transcription may constitute a developmentally regulated mechanism that modulates β enolase gene expression in skeletal muscle.

Additional copies of the actII regulatory gene induce actinorhodin production in pleiotropic bld mutants of Streptomyces coelicolor A3(2)

Summary: In S. coelicolor there are mutants (named bld) which fail to form aerial mycelium and which are also pleiotropically blocked for synthesis of several antibiotics, including the blue compound actinorhodin. Using a high copy-number vector, a DNA fragment was cloned which restored actinorhodin production, but not aerial mycelium formation, to bldA, D, F, G and H mutants. Subcloning and Southern blotting revealed that the fragment was from the region (actII) that regulates actinorhodin biosynthesis. The ability to elicit actinorhodin production and to complement an actII mutant was localized to a 1·3 kb fragment. These results indicate that the structural genes for actinorhodin biosynthetic enzymes do not contain targets for the bldA, D, F, G and H gene products, and suggest that the corresponding bld genes control actinorhodin synthesis principally via a single gene in the actII regulatory region.

Cloning and sequencing of the dnaK region of Streptomyces coelicolor A3(2)

The dnaK homologue of Streptomyces coelicolor A3(2) strain M145 has been cloned and sequenced. Nucleotide sequence analysis of a 2.5-kb region revealed an open reading frame (ORF) encoding a predicted DnaK protein of 618 amino acids (M(r) = 66,274). The dnaK coding sequence displays extreme codon bias and shows a strong preference for CGY and GGY, for Arg and Gly codons, respectively. The predicted DnaK sequence has a high Lys:Arg ratio which is not typical of streptomycete proteins. The region immediately downstream from dnaK contains an ORF for a GrpE-like protein; the predicted start codon of grpE overlaps the last two codons of dnaK, indicating that the two genes are translationally coupled. This organisation differs from that reported for other prokaryotes.