CHROMATIN STRUCTURE AND GENE-EXPRESSION

It is now well understood that chromatin structure is perturbed in the neighborhood of expressed genes. This is most obvious in the neighbor hood of promoters and enhancers, where hypersensitivity to nucleases m arks sites that no longer carry canonical nucleosomes, and to which tr anscription factors bind. To study the relationship between transcript ion factor binding and the generation of these hypersensitive regions, we mutated individual cis-acting regulatory elements within the enhan cer that lies between the chicken beta- and epsilon-globin genes. Cons tructions carrying the mutant enhancer were introduced by stable trans formation into an avian erythroid cell line. We observed that weakenin g the enhancer resulted in creation of two classes of site: those stil l completely accessible to nuclease attack and those that were complet ely blocked. This all-or-none behavior suggests a mechanism by which c hromatin structure can act to sharpen the response of developmental sy stems to changing concentrations of regulatory factors, Another proble m raised by chromatin structure concerns the establishment of boundari es between active and inactive chromatin domains. We have identified a DNA element at the 5' end of the chicken beta-globin locus, near such a boundary, that has the properties of an insulator; in test construc tions, it blocks; the action of an enhancer on a promoter when it is p laced between them. We describe the properties and partial dissection of this sequence. A third problem is posed by the continued presence o f nucleosomes on transcribed genes, which might prevent the passage of RNA polymerase. We show however, that a prokaryotic polymerase can tr anscribe through a histone octamer on a simple chromatin template. The analysis of this process reveals that an octamer is capable of transf erring from a position in front of the polymerase to one behind, witho ut ever losing its attachment to the DNA.