Regulation of the human beta-like globin genes has been intensively investigated because manipulation of the expression of these genes may permit amelioration of sickle cell disease as well as the beta-thalassemias. The human beta-globin locus is also a classic model of tissue specific gene regulation as well as the developmental regulation of a multigene locus. In recent years there has been a consensus that enhancers upstream of the genes (in the locus control region or LCR) directly interact with the genes and increase their transcription. Using a novel system of reporter gene analysis, however, the applicants have recently demonstrated that a key component of the LCR (5'HS2), as well as other enhancers, does not increase the expression level of a reporter gene to any significant extent, but instead counteracts silencing by repressive chromatin. The investigators have shown that promoter elements can counteract silencing as well as increase transcription; they have also found that characteristics of the integration site strongly influence expression level. The applicants propose that cis-acting transcriptional elements serve primarily to ensure that a gene is expressed in a given lineage, while transcription rate is the product of local chromatin structure and modulation by promoter elements. They also propose a series of experiments to test this hypothesis. The investigators will ask if addition of an enhancer to a locus will stabilize expression; if the proximity of promoter elements to the initiator determines their ability to regulate transcription rate; and if insulators, MARs, and enhancers can alter position effects on expression level. Applicants also will study the coordinate regulation of a model two-gene locus they have developed. All of these experiments will be done in K562 erythroleukemia cells, but they plan to also make ES-derived mice to ask if the 5'HS2 globin enhancer can suppress silencing of gene expression in erythroid cells, and if it can silence expression in non-erythroid cells. These studies may prove useful in the design of gene replacement strategies or drug therapies for the hemoglobinopathies, as well as improving basic knowledge of gene expression.
Showing the most recent 10 out of 20 publications
