Identification of Differentially Expressed Nucleolar TGF-β1 Target (DENTT) in Human Lung Cancer Cells That Is a New Member of the TSPY/SET/NAP-1 Superfamily

The transforming growth factor-β1 (TGF-β1) responsive epithelial non-small-cell lung cancer (NSCLC) cell line NCI-H727 was used to identify potential target genes involved in TGF-β1-mediated responses. Comparative cDNA expression patterns between cells treated with TGF-β1 and those treated with vehicle were generated by differential mRNA display. One 496-bp fragment, differentially increased threefold by TGF-β1 and hybridizing to a 2.7-kb mRNA species in NCI-H727 cells by Northern analysis, revealed no significant match to any known gene sequence. The mRNA transcript of this novel gene that we named differentially expressed nucleolar TGF-β1 target (DENTT) is expressed in several normal human tissues, with the highest level of expression in brain. Human brain cDNA library screening and 5′ rapid amplification of cDNA ends yielded full-length DENTT cDNA containing an 1899-bp open reading frame encoding a predicted 633-amino-acid protein with four potential nuclear localization signals (NLSs) and two coiled-coil regions. DENTT contains a conserved 191-residue domain that shows significant identity to, and defines, the TSPY/TSPY-like/SET/NAP-1 superfamily. Enhanced green fluorescent protein (EGFP)-tagged full-length DENTT transfected into COS-7 cells showed nucleolar and cytoplasmic localization. Transfection of EGFP-tagged DENTT NLS deletion constructs lacking the bipartite NLS-1 were excluded from the nucleolus. While NLS-1 is necessary for nucleolar localization of DENTT, it is not sufficient for sole nucleolar localization. Our data show that DENTT mRNA induction by TGF-β1 correlates with induction of TGF-β1 mRNA, induction of extracellular matrix gene expression, and inhibition of colony formation in soft agarose in TGF-β1 responsive NSCLC cells when exposed to TGF-β1. TGF-β1 does not induce DENTT mRNA expression in TGF-β1 nonresponsive NSCLC cells. Our data suggest that this novel TGF-β1 target gene has distinct domains for direction to different subnuclear locations.