UBF-binding site arrays form pseudo-NORs and sequester the RNA polymerase I transcription machinery.
Human ribosomal genes (rDNA) are located in nucleolar organizer regions (NORs) on the short arms of acrocentric chromosomes. Metaphase NORs that were transcriptionally active in the previous cell cycle appear as prominent chromosomal features termed secondary constrictions that are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I (pol I) transcription factor UBF binds extensively across rDNA throughout the cell cycle. To determine if UBF binding underpins NOR structure, we integrated large arrays of heterologous UBF-binding sequences at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus and, during metaphase, form novel silver stainable secondary constrictions, termed pseudo-NORs, morphologically similar to NORs. We demonstrate for the first time that in addition to UBF the other components of the pol I machinery are found associated with sequences across the entire human rDNA repeat. Remarkably, a significant fraction of these same pol I factors are sequestered by pseudo-NORs independent of both transcription and nucleoli. Because of the heterologous nature of the sequence employed, we infer that sequestration is mediated primarily by protein-protein interactions with UBF. These results suggest that extensive binding of UBF is responsible for formation and maintenance of the secondary constriction at active NORs. Furthermore, we propose that UBF mediates recruitment of the pol I machinery to nucleoli independently of promoter elements.
Mais
Christine
C
Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY Scotland, United Kingdom.
Wright
Jane E
JE
Prieto
José-Luis
JL
Raggett
Samantha L
SL
McStay
Brian
B
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism; D002875:Chromosomes; D004275:DNA, Ribosomal / Q000378:metabolism; D005818:Genetic Engineering; D006801:Humans; D046911:Macromolecular Substances; D008677:Metaphase; D009697:Nucleolus Organizer Region / Q000648:ultrastructure*; D035463:Pol1 Transcription Initiation Complex Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D021381:Protein Transport; D012318:RNA Polymerase I / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014981:Xenopus
D004275:DNA, Ribosomal; D046911:Macromolecular Substances; D035463:Pol1 Transcription Initiation Complex Proteins; C063844:transcription factor UBF; D012318:RNA Polymerase I