Regulation of alpha-fetoprotein expression by Nkx2.8.
The alpha-fetoprotein (AFP) gene is an important model of developmental gene silencing and neoplastic gene reactivation. Nkx2.8 is a divergent homeodomain factor originally cloned through its binding to the promoter-coupling element (PCE), a regulatory region upstream of the AFP promoter that mediates stimulation by distant enhancers. Nkx2.8 is the only developmentally regulated factor that has been associated with AFP gene expression. Fetoprotein transcription factor, an orphan nuclear receptor, has also been shown to bind the PCE but is not developmentally regulated. The binding specificities of both families of transcription factor were determined, and overlapping sites for each were defined in the PCE. After modification of nuclear extract and gel shift analysis procedures, Nkx2.8 was identified in six AFP-positive cell lines. Transient-transfection analysis did not show transcriptional stimulation by Nkx2.8 or other active NK2 factors, which only interfered with gene expression. However, two sets of analysis demonstrated the relationship of Nkx2.8 to AFP expression: chromatin immunoprecipitation demonstrated that Nkx2.8 bound to the active AFP promoter, and antisense inhibition of Nkx2.8 mRNA translation selectively reduced expression of both the endogenous human AFP gene and transfected reporters containing the rat AFP promoter.
Kajiyama
Yasuo
Y
Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Tian
Jianmin
J
Locker
Joseph
J
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic; D051717:Fushi Tarazu Transcription Factors; D005786:Gene Expression Regulation / Q000502:physiology*; D006367:HeLa Cells / Q000737:chemistry; D018398:Homeodomain Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid; D013045:Species Specificity; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000737:chemistry; D000509:alpha-Fetoproteins / Q000096:biosynthesis* / Q000235:genetics
D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; C108070:NKX2-8 protein, human; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; D000509:alpha-Fetoproteins; C099948:fetoprotein transcription factor