UBF binding in vivo is not restricted to regulatory sequences within the vertebrate ribosomal DNA repeat - PubMed (original) (raw)
UBF binding in vivo is not restricted to regulatory sequences within the vertebrate ribosomal DNA repeat
Audrey C O'Sullivan et al. Mol Cell Biol. 2002 Jan.
Abstract
The HMG box containing protein UBF binds to the promoter of vertebrate ribosomal repeats and is required for their transcription by RNA polymerase I in vitro. UBF can also bind in vitro to a variety of sequences found across the intergenic spacer in Xenopus and mammalian ribosomal DNA (rDNA) repeats. The high abundance of UBF, its colocalization with rDNA in vivo, and its DNA binding characteristics, suggest that it plays a more generalized structural role over the rDNA repeat. Until now this view has not been supported by any in vivo data. Here, we utilize chromatin immunoprecipitation from a highly enriched nucleolar chromatin fraction to show for the first time that UBF binding in vivo is not restricted to known regulatory sequences but extends across the entire intergenic spacer and transcribed region of Xenopus, human, and mouse rDNA repeats. These results are consistent with a structural role for UBF at active nucleolar organizer regions in addition to its recognized role in stable transcription complex formation at the promoter.
Figures
FIG. 1.
Size range of soluble nucleolar chromatin. DNA extracted from Xenopus (X) and human (H) soluble nucleolar chromatin was electrophoresed on a 1.2% agarose gel alongside a molecular weight ladder (M). The lengths in kilobases of key bands in the marker lane are shown on the left. Ethidium bromide-stained DNA was quantified using a Molecular Imager (Bio-Rad). Profiles of DNA extracted from Xenopus and human cell nucleolar chromatin compared to the molecular weight marker are shown below the gel.
FIG. 2.
α-xUBF antibodies recognize Xenopus NORs. Metaphase chromosome spreads (counterstained with 4′,6-diamidino-2-phenylindole [DAPI]) from Xenopus XLK2 cells were subject to combined immunofluorescence and FISH. UBF was visualized using α-xUBF antibody (FITC/green). FISH was performed with Spectrum Red-labeled Xenopus rDNA. Secondary constrictions on DAPI-stained chromosomes are indicated by arrows(a). Panels b and c show UBF staining and FISH signals, respectively. The merge of UBF and FISH signals is shown in panel d.
FIG. 3.
UBF binds to Xenopus and human ribosomal gene promoters in vivo. Quantitative PCR was performed using promoter-specific primers and DNA extracted from α-xUBF and preimmune ChIP assays. Gels from experiments with human and Xenopus nucleolar chromatin are shown in the top and bottom panels, respectively. See Materials and Methods for details of the ChIP assay, primers, and PCR conditions. Control PCRs were performed with the human rDNA repeat cosmid N68f1 and the Xenopus plasmid pXlr101A. The amount of cloned DNA used in control reaction mixtures (10 to 0.01 ng) is shown above each lane as appropriate. Concentrations of SDS and Triton X-100 used in immunoprecipitation washes are shown above.
FIG. 4.
UBF binds extensively over the Xenopus rDNA repeat. (A) The structure of the Xenopus rDNA repeat. Solid black boxes indicate 18S, 5.8S, and 28S rRNA coding sequences. Open boxes represent 5′ and 3′ external transcribed spacers and internal transcribed spacers. Clusters of vertical bars represent blocks of enhancer elements. The scale bar below represents the length of the repeat in kilobases. The position and size of subclones 1 to 10 of the Xenopus rDNA repeat are shown below. (B) Duplicate arrays of subclones 1 to 10 (slots 1 to 10) and vector controls (slots C) were arrayed and probed with radiolabeled cloned rDNA repeat (top) and DNA extracted from both preimmune (middle) and α-xUBF (bottom) ChIP assays. Relative UBF loading values are shown below.
FIG. 5.
UBF binds extensively over the human rDNA repeat. (A) The structure of the human rDNA repeat is illustrated. Solid black boxes indicate 18S, 5.8S, and 28S rRNA coding sequences. Open boxes represent 5′ and 3′ external transcribed spacers. Vertical arrows represent transcription termination elements. The cross-hatched box represents a cdc27 pseudogene. The scale bar below represents the length of the repeat, in kilobases. The position and size of subclones of the human rDNA repeat (A1 to D5) are shown below the repeat. Clones with an asterisk are devoid of alu elements. (B) Insert DNA from each of the above subclones was generated by PCR and arrayed onto nylon filters (top). Fragments derived from vector DNA (D6 to D8) provided negative controls. The identity of each insert is indicated by letters and numerals on the left and top of the array, respectively. Inserts devoid of alu elements were also arrayed independently (bottom). In panel B, the identities of each insert are shown. Arrays were hybridized with an equimolar mixture of the cosmid N68f1 and the plasmid pCD7H/B (left). In experiments shown in the middle and right panels, arrays were probed with radiolabeled DNA from α-UBF and preimmune ChIP assays, respectively. (C) The relative UBF loading values for the experiment shown above. (D) The relative UBF loading values for an independent experiment (data not shown).
FIG. 6.
PCR confirms distribution of UBF on the human rDNA repeat. PCR was performed on DNA extracted from preimmune and α-UBF ChIP assays with primer pairs from across the human rDNA repeat. The locations of the primer pairs and gels of the resulting PCRs are shown in the appropriate position below a diagram of the human rDNA repeat. The source of DNA used in each PCR is shown below the gel.
FIG. 7.
UBF does not bind to satellite DNA sequences adjacent to the NORs of human chromosomes. Individual slot blots were loaded with DNA fragments representing α-satellite sequences from 14/22 and 13/15 chromosome pairs, satellite 1, and β-satellite, a pool of _alu_-free subclones of the human rDNA repeat (see Fig.5), and an intact human rDNA repeat (cosmid N38f1). Slot blots were hybridized with radiolabeled total nucleolar chromatin (top) and DNA recovered from an α-UBF ChIP assay (bottom).
FIG. 8.
RNA Pol I and SL1 show a resticted distribution on the human rDNA repeat. (A) PCR was performed with DNA extracted from preimmune, α-UBF, and α-Pol I ChIP assays with primer pairs from across the human rDNA repeat. The locations of the primer pairs and gels of the resulting PCRs are shown in the appropriate position below a diagram of the human rDNA repeat. The source of DNA used in each PCR is shown below the gel. (B) PCR was performed with DNA extracted from α-TafI110 and α-TafI48 ChIP assays with primer pairs from across the human rDNA repeat. The identity of the primer pairs is shown above the appropriate gel lanes, and the identity of the antibody used in ChIP is shown alongside.
FIG. 9.
UBF binds to sequences across the mouse rDNA repeat. PCR was performed with DNA extracted from preimmune and α-UBF ChIP assays with primer pairs from across the mouse rDNA repeat. The locations of the primer pairs and gels of the resulting PCRs are shown in the appropriate position below a diagram of the mouse rDNA repeat. The primer pair MEn is derived from the repeated enhancer elements, depicted by the cluster of vertical lines upstream of the transcribed region. The source of DNA used in each PCR is shown below the gel.
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