G-quadruplex enabling sequences within the human Tyrosine Hydroxylase promoter differentially regulates transcription.

G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repressors of transcription. Here we report a guanosine (G)-rich segment in the 3'-proximal promoter region of human tyrosine hydroxylase (TH), which acts as a necessary element for transcription. Tyrosine hydroxylase catalyzes the rate-limiting step in the catecholamine biosynthesis and is linked to several common neurological disorders such as Parkinson's and schizophrenia. A 45 nucleotide (nt) sequence (wtTH49) within the human TH promoter contains multiple G-stretches that are extremely well conserved among the primates but deviate in rodents, which raises the possibility of variation in the GQstructures formed in the two orders with the potential for a distinctive functional outcome. Biochemical and biophysical studies, including single-molecule Forster resonance energy transfer, indicate that the wtTH49 sequence can adopt multiple GQstructures by using different combinations of G-stretches. A functional assay performed with 2.8 kb of the 3'-proximal end of the TH promoter and a mutated version (TH49fm; mutated wtTH49) that is unable to form any GQ structure indicates that overall the GQ:enabling wtTH49 sequence is functionally necessary and enhances human TH promoter activity by 5-fold compared to that of the mutant. Two additional mutants, each of which was designed to form distinct GQs, differentially affected reporter gene transcription. A cationic porphyrin TMPyP4 destabilizes the wtTH49 GQ and lowers the level of reporter gene expression, although its analogue, TMPyP2, fails to elicit any response. The 45 nt G-rich sequence within the human TH promoter can form multiple GQ structures, is a necessary element in transcription, and depending on the utilized combination of G-stretches affects transcription in different ways.