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PubMed1995

A DNA binding factor (UBF) interacts with a positive regulatory element in the promoters of genes expressed during meiosis and vegetative growth in yeast.

We have studied the bipartite regulatory element UASH/URS1 in the promoter of HOP1, whose product is required for synapsis and correct pairing of homologous chromosomes during the first meiotic division. HOP1 is transcriptionally repressed by the URS1 motif during vegetative growth and induced during meiotic prophase by the UASH motif in cooperation with the bifunctional URS1 site, which is required for full induction of HOP1. While URS1 is bound in vitro by the Buf and Ume6 repressor proteins, we demonstrate for the first time by electrophoretic mobility shift assays and interference footprinting that the UASH site interacts in vitro with a novel factor called UBF (UASH binding factor) which is present in haploid and diploid cycling, as well as sporulating cells. Point mutations in the HOP1 UASH motif abolish UBF-dependent DNA binding activity in vitro and meiotic HOP1 gene expression in vivo. Furthermore, we show that UBF binds in vitro to UASH-like sequences in the promoter regions of several meiosis-specific and non-specific genes and propose that UBF mediates gene expression through its interaction with the UASH motif in both cycling and sporulating cells.

Base Sequence↗
PubMed1997

When rDNA transcription is arrested during mitosis, UBF is still associated with non-condensed rDNA.

The mechanisms that control inactivation of ribosomal gene (rDNA) transcription during mitosis is still an open question. To investigate this fundamental question, the precise timing of mitotic arrest was established. In PtK1 cells, rDNA transcription was still active in prophase, stopped in prometaphase until early anaphase, and activated in late anaphase. Because rDNA transcription can still occur in prophase and late anaphase chromosomes, the kinetics of rDNA condensation during mitosis was questioned. The conformation of the rDNA was analyzed by electron microscopy from the G2/M transition to late anaphase in the secondary constriction, the chromosome regions where the rDNAs are clustered. Whether at transcribing or non-transcribing stages, non-condensed rDNA was observed in addition to axial condensed rDNA. Thus, the persistence of this non-condensed rDNA during inactive transcription argues in favor of the fact that mitotic inactivation is not the consequence of rDNA condensation. Analysis of the three-dimensional distribution of the rDNA transcription factor, UBF, revealed that it was similar at each stage of mitosis in the secondary constriction. In addition, the colocalization of UBF with non-condensed rDNA was demonstrated. This is the first visual evidence of the association of UBF with non-condensed rDNA. As we previously reported that the rDNA transcription machinery remained assembled during mitosis, the colocalization of rDNA fibers with UBF argues in favor of the association of the transcription machinery with certain rDNA copies even in the absence of transcription. If this hypothesis is correct, it can be assumed that condensation of rDNA as well as dissociation of the transcription machinery from rDNA cannot explain the arrest of rDNA transcription during mitosis. It is proposed that modifications of the transcription machinery occurring in prometaphase could explain the arrest of transcription, while reverse modifications in late anaphase could explain activation.

Animals↗
PubMed2000

Lymphocyte mitogenic transformation is accompanied by phosphorylation of the nucleolar transcription factor UBF.

Lymphoid cell stimulation with phytohemagglutinin, converting quiescent lymphocytes into rapidly-proliferating lymphoblast-like cells, is tightly coordinated with the transcription of ribosomal genes. Nuclear Run-On assays demonstrated that after the addition of PHA ribosomal gene activity increased rapidly during the first 40 hrs. and within 64 hrs. it reached its maximum. In contrast, UBF1 and UBF2 intracellular levels, examined in immunoblots, paralleled DNA replication/cell proliferation activity. During the first 40 hrs. following the addition of PHA, lymphocytes did not proliferate and UBFI and UBF2 levels nearly corresponded to that found in quiescent cells. However, the UBF1 and UBF2 intracellular contents increased nearly twofold in the interval from 40 to 64 hrs. Metabolic radiolabeling with 32P-orthophosphate showed that in quiescent lymphocytes the cellular pool of UBF proteins was in an underphosphorylated state. Within the first 40 hrs. after the addition of PHA the phosphorylation of underphosphorylated UBF1 and UBF2 pools predominated over the phosphorylation of very small amounts of neosynthesized UBF molecules. Subsequently, within the time period from 48 to 64 hrs. the phosphorylation of neosynthesized UBF proteins predominated. We speculate that the phosphorylation-mediated mobilization of the inactive underphosphorylated intracellular UBF pool during the G1 phase of the first cell division cycle is implicated in the ribosomal RNA synthesis acceleration and in the transformation of quiescent lymphocytes to lymphoblast-like proliferating cells.

Cell Division↗
PubMed1995

[The immunolocalization of the ribosomal gene transcription initiation factor UBF in the interphase and mitosis].

Serum P419 from a patient with rheumatoid arthritis with a high specificity immunolabeling nucleoli in various mammalian cells has been identified. On the Western blots of total cellular proteins or proteins extracted from isolated nucleoli it cross-reacted with a doublet of polypeptides of 97 and 94 kDa. That is why this serum has been concluded to recognize UBF, or RNA polymerase I-specific transcription initiation factor. It was shown that UBF remained bound to the nucleoli or nucleolus organizing regions (NORs) of mitotic chromosomes despite the level of rDNA transcription. Nevertheless, intranucleolar localization of UBF was dramatically changed after partial or complete block of rRNA synthesis. In pycnotic cells positive labeling was found within the whole nucleus and cytoplasm instead of nucleolus. In metaphase UBF molecules are unequally distributed between the particular NORs, whereas in anaphase they are uniformly allocated between the daughter cells.

Animals↗
PubMed1998

Immunohistochemical detection of ribosomal transcription factor UBF: diagnostic value in malignant specimens.

The nucleolar organizer regions (NORs) of human chromosome can be identified in interphase and mitotic cells by localization of some intrinsic components such as the associated enzyme RNA polymerase I. A new sensitive staining method for NORs is described using a specific antibody to the ribosomal transcription factor UBF. By indirect immunofluorescence and enzyme-labelling methods, NORs stained in benign and malignant cells from a variety of tissues with monospecific anti-UBF serum showed significant morphological differences which correlated well with histopathological evaluation. The number of NORs per cell in malignant preparations increased significantly. Furthermore, the staining of a NOR protein component such as UBF appears to be as sensitive as the silver-staining technique (AgNOR) and might be a better alternative for detecting ribosomal activity in malignant tissues.

Biomarkers, Tumor↗
PubMed1996

Intracellular distribution of HMG1, HMG2 and UBF change following treatment with cisplatin.

Cisplatin (CDDP) is a widely used cancer chemotherapeutic agent. CDDP forms well characterized intrastrand cross-links between adjacent purines in genomic DNA. In mammalian cells, these lesions are repaired by the nucleotide excision repair system. An early event in the recognition and processing of cis-Pt-DNA adducts may well involve the binding of specific proteins to the sites of damage. Several proteins have been identified, including high mobility group (HMG) proteins 1 and 2 and upstream binding factor (UBF), which recognize CDDP-DNA. However, the physiological significance of this binding has not been established. In this study, we have utilized antibodies to these proteins to examine the effect of CDDP on their intracellular distribution. Marked changes in the immunofluorescent staining pattern of HMG1/HMG2 were noted in cells treated with CDDP. At higher drug concentrations, the distribution of UBF also changed, from a clustered appearance associated with the nucleoli to more diffuse nuclear staining. These results demonstrate that HMG1/HMG2 and UBF respond to drug treatment, presumably by recognizing cis-Pt-DNA adduct formation in intact cells. Hence, these proteins may play an important role in directing the response of tumor cells following exposure to CDDP.

Antineoplastic Agents↗
PubMed2003

Cardiac hypertrophy in vivo is associated with increased expression of the ribosomal gene transcription factor UBF.

The ribosomal DNA transcription-specific factor, UBF, is a key target for the regulation of ribosomal RNA synthesis and hypertrophic growth of isolated neonatal cardiomyocytes. In this study, we have examined whether UBF expression is also an important determinant of cardiac growth rates in vivo. We show that rDNA transcription, rRNA synthesis and UBF expression in left ventricular myocytes isolated from mice 1-6 weeks following transverse aortic constriction were significantly increased (2.5-3.5-fold) compared to the levels in myocytes from the left ventricle of sham-operated mice.

Animals↗
PubMed2004

MAD1 and c-MYC regulate UBF and rDNA transcription during granulocyte differentiation.

The regulation of cell mass (cell growth) is often tightly coupled to the cell division cycle (cell proliferation). Ribosome biogenesis and the control of rDNA transcription through RNA polymerase I are known to be critical determinants of cell growth. Here we show that granulocytic cells deficient in the c-MYC antagonist MAD1 display increased cell volume, rDNA transcription and protein synthesis. MAD1 repressed and c-MYC activated rDNA transcription in nuclear run-on assays. Repression of rDNA transcription by MAD1 was associated with its ability to interact directly with the promoter of upstream binding factor (UBF), an rDNA regulatory factor. Conversely, c-MYC activated transcription from the UBF promoter. Using siRNA, UBF was shown to be required for c-MYC-induced rDNA transcription. These data demonstrate that MAD1 and c-MYC reciprocally regulate rDNA transcription, providing a mechanism for coordination of ribosome biogenesis and cell growth under conditions of sustained growth inhibition such as granulocyte differentiation.

Animals↗
PubMed1999

Phosphorylation by G1-specific cdk-cyclin complexes activates the nucleolar transcription factor UBF.

Transcription of rRNA genes by RNA polymerase I increases following serum stimulation of quiescent NIH 3T3 fibroblasts. To elucidate the mechanism underlying transcriptional activation during progression through the G1 phase of the cell cycle, we have analyzed the activity and phosphorylation pattern of the nucleolar transcription factor upstream binding factor (UBF). Using a combination of tryptic phosphopeptide mapping and site-directed mutagenesis, we have identified Ser484 as a direct target for cyclin-dependent kinase 4 (cdk4)-cyclin D1- and cdk2-cyclin E-directed phosphorylation. Mutation of Ser484 impairs rDNA transcription in vivo and in vitro. The data demonstrate that UBF is regulated in a cell cycle-dependent manner and suggest a link between G1 cdks-cyclins, UBF phosphorylation and rDNA transcription activation.

3T3 Cells↗
PubMed1991

Cloning and structural analysis of cDNA and the gene for mouse transcription factor UBF.

The gene and protein structure of the mouse UBF (mUBF), a transcription factor for mouse ribosomal RNA gene, have been determined by cDNA and genomic clones. The unique mUBF gene consists of 21 exons spanning over 13 kb. Two mRNAs coding for mUBF1 and mUBF2 having 765 a.a. and 728 a.a., respectively, are produced by an alternative splicing of exon 8. It specifies 37 amino acids constituting a part of the regions homologous to high mobility group proteins (HMG box 2). A human UBF (hUBF) cDNA obtained by polymerase chain reaction also indicates the presence of two kinds of mRNAs, the shorter form lacking the same region as mUBF2. Comparison of the cDNAs from hUBF and mUBF revealed an unusual conservation of nucleotide sequence in the 3'-terminal non-coding region. We examined the relative amounts of expression of mUBF1 and mUBF2. The eight tissues studied contained both molecular species, although mUBF2 was the predominant form of UBF. The mRNA of mUBF1 was expressed one half of the mUBF2 in quiescent mouse fibroblasts but reached the same amount in growing state.

Amino Acid Sequence↗
PubMed1996

Structure of recombinant rat UBF by electron image analysis and homology modelling.

We have studied the structure of recombinant rat UBF (rrUBF), an RNA polymerase I transcription factor, by electron microscopy and image analysis of single particles contrasted with methylamine tungstate. Recombinant rat UBF appeared to be a flat, U-shaped protein with a central region of low density. In the dominant projections, 2-fold mirror symmetry was seen, consistent with the dimerization properties of this molecule, and of dimensions in agreement with the length of DNA that rat UBF protects in footprinting studies. Electron microscopy of various rrUBF-DNA complexes confirmed that our recombinant protein was fully able to bind the 45S rDNA promoter, and that it caused substantial bends in the DNA. Upon extended incubation in a droplet covered by a lipid monolayer at the liquid-air interface, rrUBF formed long filamentous arrays with a railway track appearance. This structure was interpreted to consist of overlapping rrUBF dimers 3.5 nm apart, which value would represent the thickness of the protein. Our results show rrUBF to interact with and bend the promoter DNA into a roughly 10 nm diameter superhelix. Based on all these electron microscopical results, an atomic structure was predicted by homology modelling of the HMG fingers, and connected by energy minimized intervening segments.

Animals↗
PubMed1995

Activity of RNA polymerase I transcription factor UBF blocked by Rb gene product.

The protein encoded by the retinoblastoma susceptibility gene (Rb) functions as a tumour suppressor and negative growth regulator. As actively growing cells require the ongoing synthesis of ribosomal RNA, we considered that Rb might interact with the ribosomal DNA transcription apparatus. Here we report that (1) there is an accumulation of Rb protein in the nucleoli of differentiated U937 cells which correlates with inhibition of rDNA transcription; (2) addition of Rb to an in vitro transcription system inhibits transcription by RNA polymerase I; (3) this inhibition requires a functional Rb pocket; and (4) Rb specifically inhibits the activity of the RNA polymerase I transcription factor UBF (upstream binding factor) in vitro. This last observation was confirmed by affinity chromatography and immunoprecipitation, which demonstrated an interaction between Rb and UBF. These results indicate that there is an additional mechanism by which Rb suppresses cell growth, namely that Rb directly represses transcription of the rRNA genes.

Amino Acid Sequence↗
PubMed1992

Analysis of the rat ribosomal DNA promoter: characterization of linker-scanning mutants and of the binding of UBF.

To investigate the mechanism of transcription of the rat ribosomal DNA (rDNA) promoter, a series of 23 linker-scanning mutants were constructed and assayed in transfected CHO cells and with cell-free extracts. With minor variation, the results of the in vitro and in vivo assays paralleled one another. For example, these assays demonstrated that the mutagenesis of bases from -133 to -124, and those from -106 to -101 of the rDNA promoter significantly inhibited transcription both in vivo and in vitro. Both of these sites lie within the upstream promoter element (UPE) of the rDNA promoter. Several constructs, in particular one that mutated the bases between -49 and -45, were better promoters in vivo than the wild-type promoter. DNAse footprinting experiments with purified UBF, an RNA polymerase I transcription factor, demonstrated the importance of the bases between -106 and -101 for the binding of that factor, providing a positive correlation between the transcription experiments and the binding of UBF to the rDNA promoter.

Animals↗
PubMed1992

Characterization and immunolocalization of RNA polymerase I transcription factor UBF with anti-NOR serum in protozoa, higher plant and vertebrate cells.

We have used anti-NOR serum from a patient with rheumatoid arthritis, to study its reactivity on different phylogenetically separated species such as protozoa, higher plants, birds and mammals. The biochemical characteristics of the antigens detected after applying mono- and two-dimensional electrophoresis and electrophoretic transfers confirm that they correspond to the rRNA polymerase I transcription factor UBF. We have demonstrated the different molecular sizes, depending on the cell complexity, but the same neutral isoelectric points in whole cell extracts of the different species. We have also demonstrated an immunolocalization of this transcription factor to the fibrillar component in all the species studied. These results suggest a high conservation of UBF throughout evolution and the possibility of using this anti-NOR serum as a tool for the study of the structure, nucleolar organization and functional roles of the different nucleolar components.

Allium↗
PubMed1994

Immunodetection of the ribosomal transcription factor UBF at the nucleolus organizer regions of fish cells.

A human autoimmune serum to nucleolus organizer regions (NORs) has been used to localize these structures at the light microscopic level in carp and trout tissue culture cells. In interphase cells, the immunofluorescence pattern indicates that the NORs autoantigens are contained exclusively within the nucleolus of carp epithelial (EPC) and trout gonad (RTG) cells. This fluorescence is punctuate rather than uniform, and presumably represents transcriptional complexes of ribosomal DNA. During mitosis, the autoantigens are detected by immunofluorescence microscopy at the chromosomal nucleolus organizer regions of condensed chromosomes, indicating that a considerable quantity of the molecule(s) remains bound to the ribosomal RNA genes. The major nucleolus autoantigen, defined in mammals as the upstream ribosomal binding factor (UBF), has been identified on immunoblots as a 90 kDa protein in extracts from fish cell lines and tissues. Thus, NORs appear to function as nucleation centers for ribosomal RNA together with a complex set of well-conserved protein factors, such as UBF. Our results suggest evolutionary conservation from fish to mammals with respect to ribosomal RNA biosynthesis driven by RNA polymerase I.

Animals↗
PubMed1996

RNA polymerase I associated factor 53 binds to the nucleolar transcription factor UBF and functions in specific rDNA transcription.

Mouse RNA polymerase I (Pol I) has, besides its 11 bona fide subunits, three polymerase associated factors, termed PAF53, 51 and 49 with respect to the size of each molecule. In order to analyze the function of PAFs, cDNA encoding PAF53 was isolated using an oligonucleotide probe derived from an oligopeptide sequence. The cDNA of PAF53 predicts a polypeptide of 434 amino acids with a sequence similarity to yeast Pol 1 49 kDa subunit. Anti-PAF53 antibody does not block the random transcription activity of Pol I, but blocks specific transcription from mouse ribosomal RNA promoter, demonstrating the requirement of PAF53 in the accurate initiation of Pol I transcription. Moreover, PAF53 interacted with mouse UBF in vitro, as revealed by Far-Western blotting and GST pull down assays. These results, together with the accumulation of PAF53 in the nucleolus of growing cells, suggest that PAF53 is involved in the formation of the initiation complex at the promoter by mediating the interaction between Pol I and UBF for the active rRNA synthesis.

3T3 Cells↗
PubMed2006

Growth factor signaling regulates elongation of RNA polymerase I transcription in mammals via UBF phosphorylation and r-chromatin remodeling.

Synthesis of the 45S rRNA by RNA polymerase I limits cell growth. Knowledge of the mechanism of its regulation is therefore key to understanding growth control. rRNA transcription is believed to be regulated solely at initiation/promoter release. However, we found that stimulation of endogenous 45S rRNA synthesis by epidermal growth factor (EGF) and serum failed to induce an increase in RNA polymerase I engagement on the rRNA genes, despite robust enhancement of 45S rRNA synthesis. Further, endogenous transcription elongation rates were measured and found to be directly proportional to 45S rRNA synthesis. Thus, elongation is a rate-limiting step for rRNA synthesis in vivo. ERK phosphorylation of the HMG boxes of UBF, an RNA polymerase I factor essential for transcription enhancement, was shown to directly regulate elongation by inducing the remodeling of ribosomal gene chromatin. The data suggest a mechanism for coordinating the cotranscriptional assembly of preribosomal particles.

Animals↗
PubMed2001

An immediate response of ribosomal transcription to growth factor stimulation in mammals is mediated by ERK phosphorylation of UBF.

Ribosomal transcription in mammals is regulated in response to growth, differentiation, disease, and aging, but the mechanisms of this regulation have remained unresolved. We show that epidermal growth factor induces immediate, ERK1/2-dependent activation of endogenous ribosomal transcription, while inactivation of ERK1/2 causes an equally immediate reversion to the basal transcription level. ERK1/2 was found to phosphorylate the architectural transcription factor UBF at amino acids 117 and 201 within HMG boxes 1 and 2, preventing their interaction with DNA. Mutation of these sites inhibited transcription activation and abrogated the transcriptional response to ERK1/2. Thus, growth factor regulation of ribosomal transcription likely acts by a cyclic modulation of DNA architecture. The data suggest a central role for ribosome biogenesis in growth regulation.

Animals↗