In order to analyze the sequence requirements for induction of the beta-globin gene, we have developed a transient assay system in mouse erythroleukemia (MEL) cells. These cells can be chemically induced to undergo terminal differentiation during which transcription of endogenous alpha and beta globin genes is greatly increased. Our objectives was to mimic this induction effect on transiently expressed genes in order to quickly and conveniently analyze plasmid constructions with varying amounts of DNA 5' or 3' to the beta-globin promoter. We succeeded in optimizing transient assay conditions for both uninduced and induced MEL cells but observed no induction of the beta-globin gene or beta-globin fusion genes located on transfected plasmids. We have developed similar assays in parallel (project Z01 DK 25045-04) in an inducible human erythroleukemia cell line, K562, where we do detect clear effects of induction upon transiently expressed beta-globin fusion genes. We have previously shown that DNA sequences known as enhancers increased the activity of the mouse beta-globin promoter in transient assays. Enhancers are cis-acting DNA sequences which act at the level of transcription to increase gene expression. They can function in either orientation both 3' and 5' to the target gene and their level of activation is relatively independent of position. Other than two categories of short """"""""core"""""""" regions, no high degree of sequence homology among the presently identified enhancers has been observed. Our analysis of the sequences of several enhancers indicated that they contained regions of dyad symmetry. We were interested in determining if this was a common feature of enhancer sequences and, if so, whether it might suggest possible mechanisms of enhancer action. Our results indicate that while dyad symmetry is a feature of some enhancers, it does not appear to be present in others. Dyad symmetry is therefore unlikely to relate to a generalized mechanism of enhancer function, although it may play a role in the activity of enhancers that exhibit this trait.
