The expression of human small nuclear U2 RNA genes is controlled by the proximal sequence element (PSE), which determines the start site of transcription, and a distal sequence element (DSE). The DSE contains an octamer element and three Sp1 binding sites. The octamer, like the PSE, is essential for U2 transcription. The Sp1 sites contribute to full promoter activity by distance-dependent cooperative interactions with the transcription factors Sp1 and Oct-1. Here we show that purified recombinant Sp1 and Oct-1 bind cooperatively to the DSE and that they physically interact in vitro. Furthermore, we show that Sp1 and Oct-1 interact in vivo using a yeast two-hybrid system. The domain of Sp1 which interacts with Oct-1 is confined to the region necessary for transcriptional stimulation of U2 RNA transcription. This region contains the glutamine-rich activation domain B and a serine/threonine-rich part. The results demonstrate that Sp1, in addition to binding to a number of other factors, also interacts directly with transcription factor Oct-1.
The complete nucleotide sequence of a mouse mastocytoma proteoglycan core protein mRNA was determined. The mRNA, estimated to contain 1.1 kb, encodes a protein with an Mr of 16715. A 21-amino acid-residue region of the protein is composed of alternating serine and glycine residues. Southern-blot analysis of mouse genomic DNA with cDNA containing sequences corresponding to the Ser-Gly repeat region revealed more than 15 gene fragments. Hybridization with a probe corresponding to the N-terminal portion of the core protein identified two fragments, and cDNA covering the C-terminal part of the core protein and the 3' untranslated part of the mRNA hybridized to a single fragment. Antibodies against the core protein, obtained after immunization of rabbits with a fusion protein, reacted with both chondroitin sulphate proteoglycans and heparin proteoglycans produced by the tumour. In immunoblotting of a microsomal fraction from the mastocytoma, the antiserum recognized a single protein (Mr 17,000), which probably represents the core protein before glycosylation.
To localize regions in the human transcription factor Sp1, which are involved in activating transcription of the U2 snRNA gene promoter and of a TATA box gene promoter, the activation potentials of GAL4/Sp1 chimeras were analyzed in mammalian cells. In vitro mutagenesis analysis of Sp1 showed that mutation of a hydrophobic amino acid residue in glutamine-rich activation domain A impairs stimulation of transcription from the TATA box promoter, but not from the U2 promoter. Furthermore, we found that similar parts of Sp1 are involved in synergistic activation of transcription together with the SV40 enhancer and with an enhancer which binds a single type of transcription factor. This suggests that the activating mechanism of Sp1 is the same with both enhancers. Interestingly, we found that the glutamine-rich domains A and B, that stimulate transcription from the TATA box promoter were not sufficient for U2 gene activation. Stimulation of U2 transcription required amino acid residues 231-485 of Sp1, which contain the glutamine-rich domain B and a serine/threonine-rich part. Since overlapping, but non-identical parts of Sp1 are required for activation of the two promoter types, we conclude that Sp1 activates the U2 snRNA and TATA box promoters by different mechanisms.
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