U RNAs are highly abundant small nuclear RNAs involved in the processing of messenger RNA. Most U RNA genes are thought to be transcribed by RNA polymerase II (pol II). However, evidence has recently been presented that U6 RNA genes are transcribed by RNA polymerase III (pol III). In the light of these results it was surprising to find that the 5' flanking region of a mouse U6 RNA gene includes a perfect copy of the octamer sequence motif, ATTTGCAT, found in many RNA polymerase II transcription enhancer elements. In the present study we show that deletion of mouse U6 gene sequences upstream of nucleotide position -217, including the octanucleotide motif, reduces U6 transcription by 90% when assayed in Xenopus laevis oocytes, suggesting the presence of a distant control element. DNase I footprinting of the 5' flanking region of the U6 gene shows protection of the octanucleotide sequence. Moreover, the 5' flanking sequence from -217 to -315 can replace the enhancer of a human U2 RNA gene. We therefore conclude that although U6 RNA genes appear to be transcribed by pol III, they are preceeded by an enhancer-like element which can functionally substitute for the enhancer of a pol II-transcribed U RNA gene.

We have examined whether the functional synergism between transcription factors Spl and OTF-1 involves cooperativity in binding. To demonstrate cooperativity, synthetic enhancers were constructed in which Spl-binding sites were combined with various OTF-1-binding sites that differed in their binding affinities. The ability of these constructions to activate transcription from the human U2 small nuclear RNA promoter was measured. The results showed that an Spl-binding site stimulated transcription 2-fold when combined with a high-affinity binding site for OTF-1. When combined with a low-affinity OTF-1-binding site, in contrast, a 20-fold stimulation of transcription was observed. The stimulatory effect of Spl was moreover influenced by the distance between the Spl-and OTF-1-binding sites and the functional cooperation was mirrored by the cooperative formation of OTF-1-and Spl-specific protein-DNA complexes in vitro. We conclude from these results that the functional cooperation between OTF-1 and Spl involves physical interactions between the two transcription factors resulting in cooperative binding. The results thus reveal a mechanism by which Spl can modulate transcription.Studies of functional and structural interactions between mammalian transcription factors have been hampered by the complexity ofmost promoter/enhancer elements and by their functional redundancy (1-3). In the present communication, we have studied the possible interactions between the transcription factors that bind to the enhancer element ofa human U2 small nuclear RNA (snRNA) gene. U2 snRNA'genes are transcribed by RNA polymerase II (4, 5), and the transcription is dependent on a distal enhancer that functions in conjunction with a proximal sequence element (PSE), located 50-60 base pairs (bp) upstream of the cap site (6-10). It has been shown (11, 12) that the U2 enhancer includes one so called octamer motif ATGCAAAT (13) 4732The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Protein/DNA interactions in the human U2 RNA gene enhancer have been characterized by DNase I footprint and DMS methylation protection analyses. Nuclear factors present in both HeLa and B cell extracts have been shown to protect an approximately 70 bp region from DNase I digestion. DMS and DNase I footprint competition studies demonstrated that the entire footprint can be accounted for by interactions with two previously identified transcription factors. One of these recognizes the so called octanucleotide-motif ATGCAAAT (transcription factor IgNF-A) which has been shown to be essential for transcription. The other is the transcription factor Sp1 which binds to three target sequences located adjacent to the octameric motif. The Sp1 interactions appear to be required for full transcriptional activity. No differences in the DNase I footprint patterns or in the DMS methylation protections were observed when nuclear extracts from HeLa cells, two different B cell lines, or from the adenovirus-transformed 293 cell line were compared.

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We have investigated the promoter requirements for in vivo transcription of a human U4C snRNA gene following transfection into HeLa cells. Two elements required for maximal U4C transcription were identified. The first, located upstream of -50, provides a basal level of transcription 2-3% of the full activity, and probably corresponds to the previously identified snRNA gene proximal element. The distal element, centered around -220, acts as a transcriptional enhancer and contains motifs for three previously recognized transcription factors: the octamer-binding protein, NF-A, which binds to motifs in the distal elements of other snRNA genes, and two factors not previously shown to be involved in snRNA gene transcription, cAMP response element binding protein (CREB) and AP-2. The octamer and putative AP-2 motifs are required for maximal transcription of the U4C gene. Specific binding of NF-A and CREB to the motifs in the distal element has been shown in vitro by DNase I and DMS methylation protection footprint competition analyses using HeLa nuclear extracts. The presence of a binding motif for the inducible factor CREB, together with the transcriptional requirement for the putative AP-2 motif, suggests a means by which expression of snRNA genes might be regulated.