Katja Seipel

Different activation domains stimulate transcription from remote ('enhancer') and proximal ('promoter') positions.

We reported previously that the lymphocyte‐derived octamer transcription factor 2A (Oct‐2A or OTF‐2A) activated both natural immunoglobulin promoters and synthetic promoters which contain the ‘octamer’ site, but was unable by itself to stimulate transcription from a remote enhancer position. Here we examine a larger set of transcription factors with respect to their proximal versus remote activation. Since a transcription factor may contain more than one activation domain, we have chosen to study the potential of individual activation domains in the context of fusion proteins that contain the DNA binding domain of GALA. We have identified at least two distinct functional classes of transcriptional activation domains. ‘Proximal’ activation domains, exemplified by glutamine‐rich domains of Oct‐1, Oct‐2A and Sp1, stimulate transcription only from a position close to the TATA box, usually in response to a remote enhancer. ‘General’ activation domains, derived from VP16, GAL4, p65 (NF‐chi B), TFE3, ITF‐1 and ITF‐2, can activate transcription from remote as well as proximal positions. These domains contain many acidic amino acids and/or other features such as clusters of serine and threonine. The proline‐rich activation domains of AP‐2 and CTF/NF1 may represent a third class with considerable promoter activity and low but significant enhancer activity. Furthermore, activation domains of both the acidic and glutamine‐rich types seem to have a modular structure, since duplicated subdomains can substitute for the entire domain.

Functional differences between mammalian transcription activation domains at the yeast GAL1 promoter.

We have fused representatives of three structurally and functionally distinct classes of mammalian transcription activation domains for RNA polymerase II to the yeast GAL4 DNA binding domain. All fusion proteins were stable when expressed in yeast and were tested for their ability to activate transcription from various positions in the yeast GAL1 promoter. Activation domains functional from remote as well as TATA‐proximal positions in mammalian cells, e.g. the acidic‐type domain of VP16, also stimulate transcription in yeast from various promoter positions. Proline‐rich domains, as e.g. in AP‐2 and CTF/NF1, with considerable promoter activity and low enhancer activity in mammalian cells stimulate transcription in yeast only from a position close to the TATA box. The glutamine‐rich domains of Oct1, Oct2 and Sp1, which activate transcription in mammalian cells from close to the TATA box in response to a remote enhancer, are inactive in the yeast GAL1 promoter. This finding might reflect some basic difference between the organization of yeast and mammalian promoters.

Both Oct-1 and Oct-2A contain domains which can activate the ubiquitously expressed U2 snRNA genes.

The U2 snRNA genes, which are transcribed by RNA polymerase II at high levels in all tissues examined, require both a distal and a proximal sequence element for efficient expression. The distal sequence element which has many properties in common with transcriptional enhancers contains, in addition to Sp1 binding sites, an octamer binding site which mediates activation through interactions with the ubiquitous transcription factor Oct‐1. In the present study we have attempted to answer the question whether Oct‐1 contains a unique activating domain which is required for activation of snRNA genes or whether ubiquitously expressed and lymphoid specific octamer binding factors both have the capacity to activate snRNA transcription. Our results show that in the presence of Oct‐1, overexpression of Oct‐2A in HeLa or COS1 cells neither inhibits nor stimulates transcription of U2 constructions which contain octamer binding sites with or without an adjacent Sp1 binding site. Moreover, an Oct‐2A‐‐GAL4 fusion protein in which the DNA binding domain of Oct‐2A was substituted for by the one of the yeast transcription activator GAL4 activates transcription of a human U2 snRNA gene in which the octamer binding site was replaced by a GAL4 binding site. From the results it is concluded that both Oct‐1 and Oct‐2A contain domains which can activate the ubiquitously expressed U2 snRNA genes.