Cooperative binding of the Xenopus RNA polymerase I transcription factor xUBF to repetitive ribosomal gene enhancers.

Abstract

Upstream binding factor (UBF) is a DNA-binding transcription factor implicated in ribosomal gene promoter and enhancer function in vertebrates. UBF is unusual in that it has multiple DNA-binding domains with homology to high-mobility-group (HMG) nonhistone chromosomal proteins 1 and 2. However, a recognizable DNA consensus sequence for UBF binding is lacking. In this study, we have used gel retardation and DNase I footprinting to examine Xenopus UBF (xUBF) binding to Xenopus laevis ribosomal gene enhancers. We show that UBF has a minimum requirement for about 60 bp of DNA, the size of the short enhancer variant in X. laevis. Stronger UBF binding occurs on the longer enhancer variant (81 bp) and on multiple enhancers linked head to tail. In vivo, Xenopus ribosomal gene enhancers exist in blocks of 10 alternating 60- and 81-bp repeats within the intergenic spacer. In vitro, UBF binds cooperatively to probes with 10 enhancers, with five intermediate complexes observed in titration experiments. This suggests that, on average, one UBF dimer binds every two enhancers. A single UBF dimer can produce a DNase I footprint ranging in size from approximately 30 to about 115 bp on enhancer probes of different lengths. This observation is consistent with the hypothesis that multiple DNA-binding domains or subdomains within UBF bind independently, forming more-stable interactions on longer probes.

UBF-binding site arrays form pseudo-NORs and sequester the RNA polymerase I transcription machinery

“…These included their high affinity for UBF and lack of homology with the human ribosomal gene repeat. An important feature is their ability to bind UBF in a cooperative manner (Putnam and Pikaard 1992), a fact we believe was critical to the success of these experiments. We envisage that murine enhancer elements (Kuhn et al 1990) and as yet unidentified elements in the human ribosomal gene repeat bind UBF in a similar manner to XEn sequences.…”

“…UBF binds cooperatively to these Xenopus Enhancer (XEn) elements Putnam and Pikaard 1992) and is required for enhancer function (McStay et al 1997). Despite the lack of sequence homology with human rDNA, human UBF can bind to these elements McStay et al 1997).…”

The RNA polymerase I transcription factor UBF is a sequence-tolerant HMG-box protein that can recognize structured nucleic acids

“…Primer 2 was end-labelled and annealed to the 66 mer and second strand synthesis was performed using the Klenow fragment of DNA polymerase I (New England Biolabs) and all four nucleoside triphosphates (1mM). The resulting double stranded probe was gel purified and incubated (in probe excess) with UBF and subjected to gel mobility shift analysis (37). Following electrophoresis, the gel was exposed to film and the shifted UBF -probe complexes localized, excised, and eluted in 100 microliters of TE, pH 8.0.…”

“…UBF-DNA interaction conditions and probes DNase I footprinting and gel mobility shift assays (using 4% polyacrylamide, 25 mM TBE gels) involved the use of 5' endlabelled probes ( -0.2ng probe per reaction) and 5-10 ng highly purified UBF as described previously (12,37).…”

“…Xenopus UBF purification UBF was purified from isolated nuclei of an X. laevis kidney cell line (line Xlk2) by chromatography on DEAE -Sepharose, Biorex 70, and Mono Q as described (12,37). UBF-DNA interaction conditions and probes DNase I footprinting and gel mobility shift assays (using 4% polyacrylamide, 25 mM TBE gels) involved the use of 5' endlabelled probes ( -0.2ng probe per reaction) and 5-10 ng highly purified UBF as described previously (12,37).…”