A novel MASH1 enhancer with N-myc and CREB-binding sites is active in neuroblastoma.
Neuroblastoma is one of the most common solid tumors in childhood. With the aim of developing a targeting vector for neuroblastoma, we cloned and characterized an enhancer in the 5'-flanking regions of the MASH1 gene by a random-trap method from a 36 kb cosmid DNA. The enhancer-containing clone was identified by the expression of GFP when transfected into neuroblastoma cell lines. The enhancer-luciferase activity is higher in neuroblastoma cell lines, IMR32, BE2 and SH-SY5Y, compared with those in non-neuroblastoma cell lines, U1242 glioma, N417 small cell lung cancer and EOMA hemangioma. The core enhancer was determined within a 0.2 kb fragment, yielding three- to fourfold higher activity than that of the MASH1 promoter alone in IMR32 and BE2. This area possesses GATA- and CREB-binding sites, as well as the E-box. EMSA on this area demonstrated that CREB/ATF could bind the DNA. Chromatin immunoprecipitation assay revealed that N-myc, CREB, and co-activators CBP and PCAF, but not HDAC1, are bound to the core enhancer at the same time as the co-activators and N-myc bind to the promoter. This supports the idea that the commonly overexpressed genes HASH1 and N-myc are regulated in concert, confirming their importance as prognostic markers or targets for therapy.
Watt
F
F
Children's Cancer Institute Australia for Medical Research, Randwick, New South Wales, Australia.
Watanabe
R
R
Yang
W
W
Agren
N
N
Arvidsson
Y
Y
Funa
K
K
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics* / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured
D051792:Basic Helix-Loop-Helix Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D016271:Proto-Oncogene Proteins c-myc; C065500:ASCL1 protein, human; C115163:CREB1 protein, human