ANÁLISIS E INTERPRETACIÓN DE LOS RESULTADOS POR CATEGORÍA Y

While the deletion analysis suggested a positive role for the M-box, it was difficult to assess the effects of further deletions since the deletion to -100 resulted in a very low level of promoter activity. To analyse the role of sequences more 3' to position -100 we chose to examine the role of the M-box together with the adjacent CR1 in the context of the intact tyrosinase promoter. To do this, two linker-scanner mutations were constructed, LS-M and LS-CR1, affecting the conserved CATGTG motif within the M-box and CR1 respectively, and the efficiency of these mutant tyrosinase promoters compared to that of the WT after transfection into B16 melanoma cells. The locations of the mutations and their effects on tyrosinase transcription are shown in Figure 3.1 OA and B respectively. While the W T tyrosinase promoter was expressed efficiently, mutation of the M-box (LS-M) resulted in a 50-fold decrease in activity, consistent with this highly conserved element playing an essential role in tyrosinase expression.

footprinted region. A. Sequence of FP3 probe and competitors with the iocation and extent of the footprint within the tyrosinase promoter indicated. The underlined sequence, GGTGGGA, is conserved within the FP5 footprint. Lower case denotes residues not derived from the tyrosinase sequence whiie asterisks indicate mutated bases. B. Band shift using melanoma nuclear extract and FP3 probe competed with 20, 100 and 500-fold molar excess of the indicated homologous or mutant competitors. The sequence of the FP5 competitor is show in Figure 3.11 A. Identical results were obtained using HeLa cell nuclear extract (not shown).

c t a a a C T A T C C C A C T G G T G G G A T A C G A G C C A A T t Fp3 * * * * * c t a g a C T A T C C C A C T G a a t t c A T A C G A G C C A A T t Fp3.M1 * * * * c t a a a C T A T C C a a a c G G T G G G A T A C G A G C C A A T t F p3.M 2 * * * * C t a a a C T A T C C C A C T G G T G G G A c g q t A G C C A A T t Fp3.M 3

B

H

i

across the tyrosinase M-box (underlined) and adjacent CR1 is shown together with the location and extent of the FP4 footprint. Mutations, indicated by the asterisks, were introduced into either the full length tyrosinase promoter or into oligonucieotides used in band shift assays. The sequence of the USF oligonucieotide is derived from the adenovirus major late promoter. B. The M-box is essential for tyrosinase promoter function. The tyrosinase CAT reporter or two mutant derivatives, LS-M or LS-CR1, containing the mutations indicated in A within the context of the fuil length promoter extending to -300, were transfected into 8 1 6 melanoma cells and CAT activity determined. No expression was observed in JEG3 ceils (not shown). C. The tyrosinase M-box binds USF in vitro. A bandshift assay using the M-box probe and the indicated competitors together with meianoma nuciear extract. Only the bound DNA is shown. The sequence of the probe and competitors is shown in 'A'. The major complex is indicated as USF. The anti-USF antibodies used were raised either against the fuii length protein (1-310) or against residues 196-272. Normal rabbit serum (NRS) was used as a control, identical results were obtained using HeLa cell extract (not shown).

B

C

Probe: M -Box USFAb

Surprisingly, mutation of tiie conserved CR1 element resulted in at most a 3-fold decrease in promoter activity. Ttiis is in agreement with our inability to detect any CR1-binding activity in the footprinting assay and suggests that despite its high degree of conservation between species, CR1 may play only a relatively minor role in expression of the human tyrosinase gene. One possibility is that the CR1 element is important at another stage in the development of the melanocyte. Note, however, that the human CR1 element contains a single base change compared to CR1 from mouse, quail and turtle. It is possible therefore that this sequence difference would adversely affect the ability of human CR1 to function as a regulatory element.

In an attempt to correlate the requirement for the M-box to binding of a specific factor, we performed band shift assays using the M-box as a probe. The results obtained are shown in Figure 3.1 OC. The major band obtained, complex 'a', appears to correspond to binding by the bHLH factor. Thus, it is competed for by the W T M-box oligonucleotide, by a heterologous competitor (USF), containing the CACGTG E-box motif from the adenovirus major late promoter, and by an oligonucleotide containing the LS- CR1 mutation, but not by the LS-M mutant. Binding by this factor therefore correlates well with the ability of the M-box to activate transcription. Although the factor binding to the M-box could correspond to any one of the multiple bHLH factors within a cell with the potential to bind an E-box motif, we asked first whether the tyrosinase M-box binding protein was related to USF, a bHLH protein readily detectable using in vitro assays. Two anti-USF antisera, raised either against the full length protein (1-301) or against residues 196-272, were used in a band shift assay together with B16-cell nuclear extract. Both antibodies could supershift complex 'a', providing strong evidence that this complex contained the USF factor. Similar results were obtained for the minor complex 'o', which may represent a USF degradation product. Since the antibodies used were directed against different regions of the USF protein and as similar results (not shown) were obtained using antibodies directed against other regions of USF it would seem unlikely that the M-box is recognised in vitro by other bHLH factors present in crude nuclear extracts. No effect was observed using normal rabbit serum (NRS). Minor ban d 'd ' appears to be relatively non-specific, being competed for only weakly by the W T M-box while formation of complex 'b' appears to be inhibited by high levels of the LS-CR1 competitor. It is not clear whether these minor complexes contribute in any way to the activity of the human tyrosinase promoter.