Pathogenesis of Parkinson's disease and related catecholaminergic neurological disorders is closely associated with changes in the levels of tyrosine hydroxylase (TH). Therefore, investigation of the regulation of the TH gene system should assist in understanding the pathomechanisms involved in these neurological disorders. To identify regulatory domains that direct human TH expression in the central nervous system (CNS), we generated two transgenic mouse lines in which enhanced yellow fluorescent protein (EYFP) is expressed under the control of either 3.2‐kb (hTHP‐EYFP construct) human TH promoter or 3.2‐kb promoter with 2‐kb 3′‐flanking regions (hTHP‐ex3‐EYFP construct) of the TH gene. In the adult transgenic mouse brain, the hTHP‐EYFP construct directs neuron‐specific EYFP expression in various CNS areas, such as olfactory bulb, striatum, interpeduncular nucleus, cerebral cortex, hippocampus, and particularly dentate gyrus. Although these EYFP‐positive cells were identified as mature neurons, few EYFP‐positive cells were TH‐positive neurons. On the other hand, we could detect the EYFP mRNA expression in a subset of neurons in the olfactory bulb, midbrain, and cerebellum, in which expression of endogenous TH is enriched, with hTHP‐ex3‐EYFP transgenic mice. These results indicate that the 3.2‐kb sequence upstream of the TH gene is not sufficient for proper expression and that the 2‐kb sequence from the translation start site to exon 3 is necessary for expression of EYFP in a subset of catecholaminergic neurons. © 2012 Wiley Periodicals, Inc.

The transcription factor Krüppel-like factor 4 (KLF4) is highly expressed and plays an important role in maintaining stemness in embryonic stem cells (ES cells). However, how the expression of KLF4 is regulated has not been well documented. To elucidate the molecular mechanisms controlling the expression of human KLF4 (hKLF4) in ES cells, a 3685-bp DNA fragment upstream of the hKLF4 gene was isolated and the 5′-regulatory region was characterized, from which two regions highly conserved with rodents were identified. In addition, one consensus binding site for members of the TCF family (TBS) was identified within each conserved region. Tcf7l1, a member of the TCF family, is highly expressed in ES cells and has been identified as a crucial regulator of the pluripotency gene regulatory network in ES cells. Therefore, the possible transcriptional mechanisms by which Tcf7l1 regulates hKLF4 expression in ES cells were investigated. In the present study, gel retardation showed that Tcf7l1 preferentially bound to the distal TBS element. In addition, overexpression of Tcf7l1 significantly increased the expression of endogenous KLF4 mRNA in both HEK293 (low nuclear β-catenin) and HCT116 cells (high nuclear β-catenin). In both cell lines, the relative expression of KLF4 mRNA was correlated with the increased Tcf7l1 gene expression, but not with active β-catenin activities. In transient transfection assays, the hKLF4-3685 construct showed 15-fold activity compared to a deletion construct (hKLF4-1033) in HCT116 cells, while significant activity was not observed in HEK293 cells. As the full promoter construct was active only in HCT116 cells, not HEK293 cells, endogenous Tcf4 may activate promoter with active β-catenin. However, Tcf7l1 overexpression showed similar promoter activation in both cells. While Tcf7l1 stimulated the hKLF-3685 promoter by 4-6 fold, it did not stimulate the hKLF4-1033 construct at all in either of the cell lines, revealing that the Tcf7l1 responsive element must be present -3685 to -1033 upstream of the translation initiation site, which was consistent with the gel retardation data. Collectively, the present results demonstrated that distal TBS was essential for TCF-mediated transcriptional activation of the hKLF4 promoter. In addition, while TBS responds to Tcf4 in a Wnt/Aβ -catenin-dependent manner, it responded to Tcf7l1 independent of active β-catenin activity.