Cellular regulation of ribosomal DNA transcription:both rat and Xenopus UBF1 stimulate rDNA transcription in 3T3 fibroblasts

Abstract

A novel RNA polymerase I (RPI) driven reporter gene has been used to investigate the in vivo role of the architectural ribosomal transcription factor UBF in gene activation and species specificity. It is shown that the level of UBF overexpression in NIH3T3 cells leads to a proportionate increase in the activities of both reporter and endogenous ribosomal genes. Further, co-expression of UBF antisense RNA suppresses reporter gene expression. Thus, UBF is limiting for ribosomal transcription in vivo and represents a potential endogenous ribosomal gene regulator. In contrast to some in vitro studies, in vivo, the mammalian and Xenopus forms of UBF1 show an equal ability to activate a mouse RPI promoter. This activity is severely impaired in mutants compromised for either dimerization or DNA binding. Similarly, the natural UBF2 splice variant shows a severely impaired capacity to activate RPI transcription. The data strongly suggest that UBF predominantly regulates ribosomal transcription by binding to and activating the ribosomal genes, but does not eliminate a possible secondary role in titrating ribosomal gene repressors such as Rb. Consistent with the DNA folding ability and cellular abundance of the UBF, we suggest that the protein may regulate a structural transition between the potentially active and active chromatin states.

A novel role for the Pol I transcription factor UBTF in maintaining genome stability through the regulation of highly transcribed Pol II genes

“…UBTF2 is inactive in regulating rDNA transcription compared with UBTF1 (Grummt 1999;Hannan et al 1999;Stefanovsky and Moss 2008) and does not function in remodeling chromatin at the rDNA repeats in the absence of UBTF1 (Sanij et al 2008). To address the relative contribution of UBTF1 and UBTF2 isoforms to Pol II histone gene expression, we performed replacement experiments with rat FLAG-Ubtf1 and FLAG-Ubtf2 cDNAs, which contain nucleic acid sequence differences in the region targeted by the murine-specific sirUbtf1/2 (Supplemental Fig.…”

RNA polymerase I transcription in confluent cells: Rb downregulates rDNA transcription during confluence-induced cell cycle arrest

“…Similar results were obtained using con¯uent 3T6 cell cultures (data not shown). Thus, in 3T6 cells, as in primary cultures of cardiomyocytes (Hannan et al, 1996a(Hannan et al, , 1999b, the overexpression of UBF1 drives rDNA expression. When 3T6 cells were co-transfected with pSMECAT, pCDNA3UBF1, and increasing amounts of pmRb (a vector which drives the expression of mouse Rb) the UBF-dependent activation of rDNA transcription was abolished (lanes 4 ± 7).…”

“…Puri®ed UBF consists of two polypeptides UBF1 (97 kD) and UBF2 (94 kD). UBF1 has been shown to activate rDNA gene transcription in vitro (Smith et al, 1990;O'Mahony and Rothblum, 1991;Paule 1994;Moss and Stefanovsky, 1995;Kuhn et al, 1994) and in vivo (Hannan et al, 1996a(Hannan et al, , 1999b. Interestingly, UBF has been shown to interact with SL-1, the A/B pocket of the retinoblastoma protein, and several subunits of RNA polymerase I (Schnapp et al, 1994;Beckman et al, 1995;Cavanaugh et al, 1995;Hanada et al, 1996;Hempel et al, 1996).…”

“…In addition, the overexpression of UBF1 in cardiomyocytes or immortalized ®broblasts drives transcription from an rDNA promoter reporter (Hannan et al, 1996a(Hannan et al, , 1999b.…”

Mass spectrometric identification of phosphorylation sites of rRNA transcription factor upstream binding factor

“…While not a novel model of rRNA transcription (12), results from the in vivo transfection assay may have uncovered unique roles for different functional regions of UBF. Specifically, mutation of serine 584 to alanine decreased transcription from the reporter construct, but had little effect in vitro.…”