Limitation in use of heterologous reporter genes for gene promoter analysis. Silencer activity associated with the cloramphenicol acetyltransferase reporter gene

Various heterologous reporter genes have been widely used for the functional characterization of gene promoters. Many such studies often found weak to very strong silencer activities to be associated with specific parts of the basal promoter or further upstream regions. In this study, we carried out a systematic study on human blood coagulation factor IX (hFIX) and anti-coagulant protein C (hPC) genes, previously shown to have silencer activities associated with their 5'-flanking regions containing promoter sequences. With newly constructed chloramphenicol acetyltransferase (CAT) reporter vectors carrying hFIX or hPC gene promoter sequences, we confirmed the strong silencer activities associated with the regions nt -1895 through nt -416 of the hFIX gene or with the region nt -802 through nt -82 of the hPC gene. However, no such silencer activities associated with the specific regions were found when autologous hFIX cDNA, hFIX minigenes, or hPC minigenes were used as reporters in the expression vector system. Relative levels of CAT, hFIX, and hPC proteins produced in the transient assays correlated well with their mRNA levels. Human FIX minigene constructs containing a simian virus 40 (SV40) 3'-untranslated region (UTR) taken from the CAT reporter gene showed no silencer activity, indicating that SV40 3'-UTR sequence of the CAT reporter gene does not contribute to the silencer activity. Expression vectors constructed with the beta-galactosidase gene under the control of hFIX gene promoter sequences also showed no silencer activity associated with the region nt -1895 through nt -416. These findings indicate that silencer activities associated with specific regions of promoter sequences as analyzed with CAT reporter genes may represent artifacts specific to the CAT reporter genes. Our findings strongly suggest a need for re-examination of promoter characterizations of many eukaryotic genes, which have been studied to date with CAT reporter genes.     

Developmental regulation of human gamma-globin genes in transgenic mice.

We report results showing that several gamma gene promoter elements participate in the developmental control of gamma-globin genes. Four gamma gene constructs with 5' truncated at -141, -201, -382, and -730 of the A gamma gene promoter linked to a micro locus control region (microLCR) cassette were used for production of transgenic mice and analysis of gamma gene expression during development. Mice carrying a microLCR -141 A gamma construct displayed downregulation of gamma gene expression in the adult stage of development, indicating that the proximal promoter contains elements participating in gamma gene silencing. Mice carrying a microLCR -201 A gamma or a microLCR -382 A gamma construct displayed high gamma gene expression in the fetal stage of development and complete loss of gamma gene downregulation in the adult stage, suggesting that the -141 to -201 gamma gene sequence contains elements which upregulate gamma gene expression and are dominant over the negative element 3' to -141. Extension of the promoter to -730 resulted in reappearance of gamma gene downregulation, suggesting that the -382 to -730 sequences contain an adult-stage-specific silencer. gamma gene expression in the microLCR -201 A gamma and the microLCR -382 A gamma transgenic mice was copy number dependent. All the microLCR -730 A gamma transgenic mice expressed gamma mRNA; however, gamma gene expression was copy number independent, indicating that levels of gamma gene expression were modulated by the surrounding chromatin. Our results suggest that multiple elements participate in gamma gene silencing. The findings in the microLCR-201 A gamma and microLCR -382 A gamma transgenic mice are interpreted to indicate that the LCR interacts not only with the minimal gamma gene promoter but also with sequences of the upstream promoter. We postulate that gamma gene downregulation is achieved when the interaction between LCR and the upstream promoter is disturbed by the silencer located in the -382 to -730 region. We propose that gamma gene silencing is achieved by the combined effect of negative elements located 3' to -141, the negative element located between -382 and -730, and the competition by the beta gene promoter during the adult stage of development.