Transcription and epigenetic profile of the promoter, first exon and first intron of the human tyrosine hydroxylase gene.
The transcriptional and chromatin profile of the promoter, first exon and first intron of the human TH gene were analyzed in human neuroblastoma BE(2)-C-16 and human renal carcinoma 293FT cell lines. The latter is a cell culture system that is not permissive for TH gene expression, whereas the former has a 50% cell fraction that tests positive for TH. The engineering of a 6.3 kb recombinant human TH promoter revealed the presence of repressors of transcription between positions (-6,244/-194). The addition of a 1.2 kb fragment of the first intron of the human TH gene (+730/+1,653) enhanced transcriptional activity of the recombinant promoter. However, both constructs were not specific for TH-positive BE(2)-C-16 cells. Chromatin immunoprecipitation (Chip) analysis was carried out on BE(2)-C-16 and 293FT cells to probe sequences of promoter, first exon and first intron of the human TH gene from position (-448/+1,204). The presence of nucleosomes was observed approximately from position (-20/+473) in both cell lines. Chip analysis was then conducted to determine the acetylation of various lysine residues of H3 and H4 in both cell lines. All analyzed lysine residues of H3 and H4 were acetylated in BE(2)-C-16 cells, whereas 293FT cells tested positive for acetylation only in the external lysine residues of the histone tail. Our data are compatible with an active TH gene expression in a 50% cell fraction of BE(2)-C-16 cells. Further analysis of epigenetic programming might lead to the identification of the factors that determine TH gene expression specifically in dopaminergic neurons.
Romano
Gaetano
G
Department of Neurosurgery, Temple University, Philadelphia, Pennsylvania, USA.
Macaluso
Marcella
M
Lucchetti
Chiara
C
Iacovitti
Lorraine
L
D000107:Acetylation; D045744:Cell Line, Tumor; D042002:Chromatin Assembly and Disassembly; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D044127:Epigenesis, Genetic; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007438:Introns; D007668:Kidney / Q000201:enzymology / Q000473:pathology; D008239:Lysine / Q000378:metabolism; D009474:Neurons / Q000201:enzymology* / Q000473:pathology; D009707:Nucleosomes / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics* / Q000378:metabolism
D009707:Nucleosomes; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D014446:Tyrosine 3-Monooxygenase; D008239:Lysine