Agata Giallongo

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.

The PVT-1 oncogene is a myc protein target that is overexpressed in transformed cells

The human PVT‐1 gene is located on chromosome 8 telomeric to the c‐Myc gene and it is frequently involved in the translocations occurring in variant Burkitts lymphomas and murine plasmacytomas. It has been proposed that PVT‐1 regulates c‐Myc gene transcription over a long distance. To get new insights into the functional relationships between the two genes, we have investigated PVT‐1 and c‐Myc expression in normal human tissues and in transformed cells. Our findings indicate that PVT‐1 expression is restricted to a relative low number of normal tissues compared to the wide distribution of c‐Myc mRNA, whereas the gene is highly expressed in many transformed cell types including neuroblastoma cells that do not express c‐Myc. Reporter gene assays were used to dissect the PVT‐1 promoter and to identify the region responsible for the elevated expression observed in transformed cells. This region contains two putative binding sites for Myc proteins. The results of transfection experiments in RAT1‐MycER cells and chromatin immunoprecipitation (ChIP) assays in proliferating and differentiated neuroblastoma cells indicate that PVT‐1 is a downstream target of Myc proteins. J. Cell. Physiol. 213: 511–518, 2007.

An integrated humoral and cellular response is elicited in pancreatic cancer by α-enolase, a novel pancreatic ductal adenocarcinoma-associated antigen†

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with a very poor 5‐year survival rate. α‐Enolase is a glycolytic enzyme that also acts as a surface plasminogen receptor. We find that it is overexpressed in PDAC and present on the cell surface of PDAC cell lines. The clinical correlation of its expression with tumor status has been reported for lung and hepatocellular carcinoma. We have previously demonstrated that sera from PDAC patients contain IgG autoantibodies to α‐enolase. The present work was intended to assess the ability of α‐enolase to induce antigen‐specific T cell responses. We show that α‐enolase‐pulsed dendritic cells (DC) specifically stimulate healthy autologous T cells to proliferate, secrete IFN‐γ and lyse PDAC cells but not normal cells. In vivo, α‐enolase‐specific T cells inhibited the growth of PDAC cells in immunodeficient mice. In 8 out of 12 PDAC patients with circulating IgG to α‐enolase, the existence of α‐enolase‐specific T cells was also demonstrated. Taken as a whole, these results indicate that α‐enolase elicits a PDAC‐specific, integrated humoral and cellular response. It is thus a promising and clinically relevant molecular target candidate for immunotherapeutic approaches as new adjuvants to conventional treatments in pancreatic cancer.

Regulation of Murine Cytochrome c Oxidase Vb Gene Expression during Myogenesis YY-1 AND HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN D-LIKE PROTEIN (JKTBP1) RECIPROCALLY REGULATE TRANSCRIPTION ACTIVITY BY PHYSICAL INTERACTION WITH THE BERF-1/ZBP-89 FACTOR

A transcription suppressor element (sequence –481 to –320) containing a G-rich motif (designated GTG) and a newly identified CAT-rich motif (designated CATR) was previously shown to modulate expression of the mouse cytochrome c oxidase Vb gene during myogenesis. Here, we show that the GTG element is critical for transcription activation in both undifferentiated and differentiated myocytes. Mutations of the CATR motif abolished transcription repression in myoblasts while limiting transcription activation in differentiated myotubes, suggesting contrasting functional attributes of this DNA motif at different stages of myogenesis. Results show that the activity of the transcription suppressor motif is modulated by an orchestrated interplay between ubiquitous transcription factors: ZBP-89, YY-1, and a member of the heterogeneous nuclear ribonucleoprotein D-like protein (also known as JKTBP1) family. In undifferentiated muscle cells, GTG motif-bound ZBP-89 physically and functionally interacted with CATR motif-bound YY-1 to mediate transcription repression. In differentiated myotubes, heterogeneous nuclear ribonucleoprotein D-like protein/JKTBP1 bound to the CATR motif exclusive of YY-1 and interacted with ZBP-89 in attenuating repressor activity, leading to transcription activation. Our results show a novel mechanism of protein factor switching in transcription regulation of the cytochrome c oxidase Vb gene during myogenesis.