Tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2 are the enzymes involved in melanin biosynthesis and are preferentially expressed in pigment cells. Their human gene promoters share the 11-base pair M box containing a CATGTG motif, which was shown here to be bound in vitro by microphthalmia-associated transcription factor (MITF). Transient cotransfection analysis showed that MITF overexpression increased the expression of a reporter gene under the control of the human tyrosinase or TRP-1 gene promoter but not the TRP-2 promoter. The promoter activation caused by MITF is dependent on each CATGTG motif of the distal enhancer element, the M box, and the initiator E box of the tyrosinase gene and the TRP-1 M box. Furthermore, a truncated MITF lacking the carboxyl-terminal 125 amino acid residues transactivated the tyrosinase promoter less efficiently than did MITF, suggesting that MITF's carboxyl terminus contains a transcriptional activation domain, but unexpectedly such a truncated MITF remarkably transactivated the TRP-2 gene promoter. These results suggest that MITF is sufficient to direct pigment cell-specific transcription of the tyrosinase and TRP-1 genes but not the TRP-2 gene.

Microphthalmia-associated transcription factor (Mitf) plays a critical role in the development of neural crest-derived melanocytes. Here, we show that exogenously added Wnt-3a protein, an intercellular signaling molecule, up-regulates the expression of endogenous melanocyte-specific Mitf (Mitf-M) mRNA in cultured melanocytes. The melanocyte-specific promoter of the humanMITF gene (MITF-M promoter) contains a functional LEF-1-binding site, which is bound in vitro by LEF-1 and confers the preferential expression on a reporter gene in melanocytes and melanoma cells, as judged by the transient transfection assays. Moreover, the LEF-1-binding site is required for the transactivation of a reporter gene by LEF-1, β-catenin, or their combination. Exogenously added Wnt-3a protein also transactivates theMITF-M promoter via the LEF-1-binding site; this activation was abolished when a dominant-negative form of LEF-1 was coexpressed. These results suggest that Wnt-3a signaling recruits β-catenin and LEF-1 to the LEF-1-binding site of the MITF-M promoter. Therefore, the present study identifies Mitf-M/MITF-M as a direct target of Wnt signaling.

Heme oxygenase 1 (HO-1) catalyzes heme breakdown, eventually releasing iron, carbon monoxide, and bilirubin IXα. HO-1 is induced by its substrate heme and various environmental factors, which represents a protective response against oxidative stresses. Here we show that hypoxia represses HO-1 expression in three human cell types but induces it in rat, bovine, and monkey cells, indicating the inter-species difference in the hypoxic regulation of HO-1 expression. The hypoxia-mediated repression of HO-1 expression is consistently associated with the induction of Bach1, a heme-regulated transcriptional repressor, in human cells. Bach1 is a basic leucine zipper protein, forming a heterodimer with a small Maf protein. Expression of HO-1 was also reduced in human cells when exposed to interferon-γ or an iron chelator desferrioxamine, each of which induced Bach1 expression. In contrast, induction of HO-1 expression by CoCl₂ is associated with reduced expression of Bach1 mRNA. Thus, expression of HO-1 and Bach1 is inversely regulated. We have identified a Maf recognition element in the human HO-1gene that is required for repression of a reporter gene by hypoxia and targeted by Bach1. Therefore, Bach1 functions as a hypoxia-inducible repressor for the HO-1 gene, thereby contributing to fine-tuning of oxygen homeostasis in human cells.

Wnt signals regulate differentiation of neural crest cells through the β‐catenin associated with a nuclear mediator of the lymphoid‐enhancing factor 1 (LEF‐1)/T‐cell factors (TCFs) family. Here we show the interaction between the basic helix–loop–helix and leucine‐zipper region of microphthalmia‐associated transcription factor (MITF) and LEF‐1. MITF is essential for melanocyte differentiation and its heterozygous mutations cause auditory–pigmentary syndromes. Functional cooperation of MITF with LEF‐1 results in synergistic transactivation of the dopachrome tautomerase (DCT) gene promoter, an early melanoblast marker. This activation depends on the separate cis‐acting elements, which are also responsible for the induction of the DCT promoter by lithium chloride that mimics Wnt signaling. β‐catenin is required for efficient transactivation, but dispensable for the interaction between MITF and LEF‐1. The interaction with MITF is unique to LEF‐1 and not detectable with TCF‐1. LEF‐1 also cooperates with the MITF‐related proteins, such as TFE3, to transactivate the DCT promoter. This study therefore suggests that the MITF/TFE3 family is a new class of nuclear modulators for LEF‐1, which may ensure efficient propagation of Wnt signals in many types of cells.