Dysregulation of RNA polymerase I transcription during disease.

Transcription of the ribosomal RNA genes by the dedicated RNA polymerase I enzyme and subsequent processing of the ribosomal RNA are fundamental control steps in the synthesis of functional ribosomes. Dysregulation of Pol I transcription and ribosome biogenesis is linked to the etiology of a broad range of human diseases. Diseases caused by loss of function mutations in the molecular constituents of the ribosome, or factors intimately associated with RNA polymerase I transcription and processing are collectively termed ribosomopathies. Ribosomopathies are generally rare and treatment options are extremely limited tending to be more palliative than curative. Other more common diseases are associated with profound changes in cellular growth such as cardiac hypertrophy, atrophy or cancer. In contrast to ribosomopathies, altered RNA polymerase I transcriptional activity in these diseases largely results from dysregulated upstream oncogenic pathways or by direct modulation by oncogenes or tumor suppressors at the level of the RNA polymerase I transcription apparatus itself. Ribosomopathies associated with mutations in ribosomal proteins and ribosomal RNA processing or assembly factors have been covered by recent excellent reviews. In contrast, here we review our current knowledge of human diseases specifically associated with dysregulation of RNA polymerase I transcription and its associated regulatory apparatus, including some cases where this dysregulation is directly causative in disease. We will also provide insight into and discussion of possible therapeutic approaches to treat patients with dysregulated RNA polymerase I transcription. This article is part of a Special Issue entitled: Transcription by Odd Pols.

Kidney injury molecule-1 expression in transplant biopsies is a sensitive measure of cell injury.

Kidney injury molecule-1 (KIM-1) is a specific histological biomarker for diagnosing early tubular injury on renal biopsies. In this study, KIM-1 expression was quantitated in renal transplant biopsies by immunohistochemistry and correlated with renal function. None of the 25 protocol biopsies showed detectable tubular injury on histologic examination, yet 28% had focal positive KIM-1 expression. Proximal tubule KIM-1 expression was present in all biopsies from patients with histological changes showing acute tubular damage and deterioration of kidney function. In this group, higher KIM-1 staining predicted a better outcome with improved blood urea nitrogen (BUN), serum creatinine, and estimated glomerular filtration rate (eGFR) over an ensuing 18 months. KIM-1 was expressed focally in affected tubules in 92% of kidney biopsies from patients with acute cellular rejection. By contrast, there was little positive staining for Ki-67, a cell proliferation marker, in any of the groups. KIM-1 expression significantly correlated with serum creatinine and BUN, and inversely with the eGFR on the biopsy day. Our study shows that KIM-1 staining sensitively and specifically identified proximal tubular injury and correlated with the degree of renal dysfunction. KIM-1 expression is more sensitive than histology for detecting early tubular injury, and its level of expression in transplant biopsies may indicate the potential for recovery of kidney function.

Purification and characterization of a high-mobility-group-like DNA-binding protein that stimulates rRNA synthesis in vitro.

A 16,000-dalton, high-mobility-group-like (HMG-like) DNA-binding protein, referred to as p16, has been purified to homogeneity from Novikoff hepatoma ascites cells. p16 binds specifically to a portion of the 5' flanking region of the rat rRNA gene (-620 to -417), which is part of the upstream activator sequence identified previously (B. G. Cassidy, H.-F. Yang-Yen, and L. I. Rothblum, Mol. Cell. Biol. 6:2766-2773, 1986). p16 also binds to a segment of the external transcribed spacer (+352 to +545). In vitro reconstituted transcription experiments demonstrated that the addition of p16 stimulated rRNA synthesis up to ca. fourfold. The stimulation was dose dependent and saturable. The effect of p16 on ribosomal gene transcription was also dependent on the presence of either the upstream or the downstream DNA-binding site, or both. The amino acid composition of p16 is very similar to that of HMG-I, suggesting that p16 may be a member of the HMG-I family of proteins. In this case, our results suggest that HMG proteins may play an important role in the regulation of the rRNA gene expression.

Domains of the rat rDNA promoter must be aligned stereospecifically.

Efficient transcription from the rat rDNA promoter results from an undefined interaction between the core (CPE) and upstream (UPE) promoter elements or the protein complexes which form on them. These interactions were demonstrated by the behavior of promoters that contained either linker-scanning or deletion mutations of the UPE in combination with point mutations of the CPE (bidomain mutants). In vivo transcription experiments using point mutations within the CPE (G----A mutation at either -16 or -7) demonstrated that the CPE may in fact consist of two domains. Whereas both of these mutants were rescued by the addition of UBF to in vitro transcription reactions, the CPE mutant -7A/G was inactive in vivo. Experiments with these bidomain mutants demonstrated that the UPE was required for the rescue of the CPE mutants. We also examined the hypothesis that this interaction might require a stereospecific alignment of the promoter elements. Our results indicate that the promoter consists of several domains with differing responses to mutations that alter the distance between, or within, the promoter elements. For example, the insertion or deletion of half-multiples of the helical repeat distance between -167 and -147 had no significant effect on transcription. On the other hand, some sites were sensitive to deletions of any size but not to insertions of up to 20 bp. The analyses of two sites yielded results suggesting that they lay between domains of the promoter that must be on the same side of the DNA helix for promoter activity. The first of these sites mapped between -106 and -95.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional role for the nontranscribed spacer of rat ribosomal DNA.

In vitro transcription of the rat rRNA gene led to the identification of a region within a 3.4-kilobase fragment of the nontranscribed spacer (NTS) which significantly increased the transcription of rat ribosomal DNA. Promoter constructs containing this region were transcribed up to 17-fold more efficiently in vitro than templates with only 167 or 286 base pairs of NTS. This effect was also observed when the 3.4-kb fragment of the NTS was subcloned in the opposite orientation and 4 kb upstream of the promoter. The region responsible for the enhanced level of transcription was found between -286 and -1018. The results of order-of-addition experiments suggested that the enhanced level of transcription was the result of the formation of a stable complex between a trans-acting factor and the nontranscribed spacer. DNA-protein binding assays demonstrated that the same region of the NTS determined to have enhancer activity also specifically bound a proteinase K-sensitive factor present in nuclear extracts. The sequence of this region was not found to have any significant homology with the promoter of the rat rRNA gene. This is the first report to assign a transcriptional role to the NTS of a mammalian rRNA gene.

Additional RNA polymerase I initiation site within the nontranscribed spacer region of the rat rRNA gene.

We identified and characterized an additional promoter within the nontranscribed spacer (NTS) of the rat ribosomal gene repeat that is capable of supporting initiation of transcription by RNA polymerase I in vitro. Within this promoter there is a sequence of 13 nucleotides which is 100% homologous to nucleotides -18 to -6 (+1 being the first nucleotide of 45S rRNA) of the major promoter of 45S pre-rRNA and is located between nucleotides -731 and -719. To identify the exact location of the upstream initiation site, the RNA synthesized in vitro from this new promoter was gel isolated and subjected to fingerprint analysis, Southern hybridization, and reverse transcriptase elongation. Based on these analyses, the in vitro-synthesized RNA initiates with an A at nucleotide -713. When compared individually, the upstream promoter was transcribed ninefold less efficiently than the major promoter. When templates which contain both promoters on the same piece of DNA were transcribed, the major promoter was at least 50-fold more efficient.

The species-specific RNA polymerase I transcription factor SL-1 binds to upstream binding factor.

Transcription of the 45S rRNA genes is carried out by RNA polymerase I and at least two trans-acting factors, upstream binding factor (UBF) and SL-1. We have examined the hypothesis that SL-1 and UBF interact. Coimmunoprecipitation studies using an antibody to UBF demonstrated that TATA-binding protein, a subunit of SL-1, associates with UBF in the absence of DNA. Inclusion of the detergents sodium dodecyl sulfate and deoxycholate disrupted this interaction. In addition, partially purified UBF from rat cell nuclear extracts and partially purified SL-1 from human cells coimmunoprecipitated with the anti-UBF antibody after mixing, indicating that the UBF-SL-1 complex can re-form. Treatment of UBF-depleted extracts with the anti-UBF antibody depleted the extracts of SL-1 activity only if UBF was added to the extract prior to the immunodepletion reaction. Furthermore, SL-1 activity could be recovered in the immunoprecipitate. Interestingly, these immunoprecipitates did not contain RNA polymerase I, as a monospecific antibody to the 194-kDa subunit of RNA polymerase I failed to detect that subunit in the immunoprecipitates. Treatment of N1S1 cell extracts with the anti-UBF antibody depleted the extracts of SL-1 activity but not TFIIIB activity, suggesting that the binding of UBF to SL-1 is specific and not solely mediated by an interaction between UBF and TATA-binding protein, which is also a component of TFIIIB. These data provide evidence that UBF and SL-1 interact.

Characterization of factors that direct transcription of rat ribosomal DNA.

The protein components that direct and activate accurate transcription by rat RNA polymerase I were studied in extracts of Novikoff hepatoma ascites cells. A minimum of at least two components, besides RNA polymerase I, that are necessary for efficient utilization of templates were identified. The first factor, rat SL-1, is required for species-specific recognition of the rat RNA polymerase I promoter and may be sufficient to direct transcription by pure RNA polymerase I. Rat SL-1 directed the transcription of templates deleted to -31, the 5' boundary of the core promoter element (+1 being the transcription initiation site). The second factor, rUBF, increased the efficiency of template utilization. Transcription of deletion mutants indicated that the 5' boundary of the domain required for rUBF lay between -137 and -127. Experiments using block substitution mutants confirmed and extended these observations. Transcription experiments using those mutants demonstrated that two regions within the upstream promoter element were required for optimal levels of transcription in vitro. The first region was centered on nucleotides -129 and -124. The 5' boundary of the second domain mapped to between nucleotides -106 and -101. DNase footprint experiments using highly purified rUBF indicated that rUBF bound between -130 and -50. However, mutation of nucleotides -129 and -124 did not affect the rUBF footprint. These results indicate that basal levels of transcription by RNA polymerase I may require only SL-1 and the core promoter element. However, higher transcription levels are mediated by additional interactions of rUBF, and possibly SL-1, bound to distal promoter elements.

Nucleologenesis: U3 snRNA-containing prenucleolar bodies move to sites of active pre-rRNA transcription after mitosis.

We have investigated the distribution of U3 snRNA and rRNA in HeLa cells and normal rat kidney cells during interphase and mitosis. U3 snRNA, known to be involved in pre-rRNA processing, was detected in nucleoli and coiled bodies during interphase, whereas rRNA was distributed in the nucleoli and throughout the cytoplasm. By comparison, ribosomal protein S6 was detected in nucleoli, coiled bodies, and in the cytoplasm. During nucleologenesis, pre-rRNA was observed in newly forming nucleoli during late telophase but not in prenucleolar bodies (PNBs), whereas U3 snRNA was detected in forming nucleoli and PNBs. Similar findings to those reported here for the localization of U3 snRNA have been reported previously for the U3 small nuclear ribonucleoprotein fibrillarin. These results suggest that components involved in pre-rRNA processing localize to discrete PNBs at the end of mitosis. The nucleolus is formed at specific telophase domains (nucleolar organizing regions) and the PNBs, containing factors essential for pre-rRNA processing, are recruited to these sites of rRNA transcription and processing.

The role of acetylation in rDNA transcription.

Treatment of NIH 3T3 cells with trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), resulted in a dose-dependent increase in transcription from a rDNA reporter and from endogenous rRNA genes. Chromatin immunoprecipitation using anti-acetyl-histone H4 antibodies demonstrated a direct effect of TSA on the acetylation state of the ribosomal chromatin. TSA did not reverse inhibition of transcription from the rDNA reporter by retinoblastoma (Rb) protein, suggesting that the main mechanism by which Rb blocks rDNA transcription may not involve recruitment of deacetylases to rDNA chromatin. Overexpression of histone transacetylases p300, CBP and PCAF stimulated transcription in transfected NIH 3T3 cells. Recombinant p300, but not PCAF, stimulated rDNA transcription in vitro in the absence of nucleosomes, suggesting that the stimulation of rDNA transcription by TSA might have a chromatin-independent component. We found that the rDNA transcription factor UBF was acetylated in vivo. Finally, we also demonstrated the nucleolar localization of CBP. Our results suggest that the organization of ribosomal chromatin of higher eukaryotes is not static and that acetylation may be involved in affecting these dynamic changes directly through histone acetylation and/or through acetylation of UBF or one of the other components of rDNA transcription.

Isolation and characterization of complementary DNA to proliferating cell nucleolar antigen P40.

Proliferating cell nucleolar antigen P40 is a late G1-specific protein, which was found in a variety of human tumors (A. Chatterjee, J. W. Freeman, and H. Busch. Cancer Res., 47: 1123-1129, 1987). Two overlapping complementary DNA clones for antigen P40 were isolated by immunoscreening a lambda gt11 human expression library. The complete nucleotide sequence of the clones was determined. The complementary DNAs encode the Mr 30,000 portion of the COOH-terminal portion of the protein. The mRNA for P40 was 2.8 kilobases long and was expressed maximally in G1 cells in cell cycle. A series of deletion mutants of the expressed peptide was constructed and the deletion mutants were expressed in Escherichia coli. Using these mutants, the epitope region of P40 recognized by a P40-specific monoclonal antibody was identified. The hydropathy plot based on the protein sequence revealed that this region of the protein is largely hydrophilic. This protein is unique and differs in sequence from other proliferating cell nuclear/nucleolar antigen proteins of similar molecular weight such as protein B23 and cyclin.

Rb and p130 regulate RNA polymerase I transcription: Rb disrupts the interaction between UBF and SL-1.

We have previously demonstrated that the protein encoded by the retinoblastoma susceptibility gene (Rb) functions as a regulator of transcription by RNA polymerase I (rDNA transcription) by inhibiting UBF-mediated transcription. In the present study, we have examined the mechanism by which Rb represses UBF-dependent rDNA transcription and determined if other Rb-like proteins have similar effects. We demonstrate that authentic or recombinant UBF and Rb interact directly and this requires a functional A/B pocket. DNase footprinting and band-shift assays demonstrated that the interaction between Rb and UBF does not inhibit the binding of UBF to DNA. However, the formation of an UBF/Rb complex does block the interaction of UBF with SL-1, as indicated by using the 48 kDa subunit as a marker for SL-1. Additional evidence is presented that another pocket protein, p130 but not p107, can be found in a complex with UBF. Interestingly, the cellular content of p130 inversely correlated with the rate of rDNA transcription in two physiological systems, and overexpression of p130 inhibited rDNA transcription. These results suggest that p130 may regulate rDNA transcription in a similar manner to Rb.

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

When 3T6 cells are confluent, they withdraw from the cell cycle. Concomitant with cell cycle arrest a significant reduction in RNA polymerase I transcription (80% decrease at 100% confluence) is observed. In the present study, we examined mechanism(s) through which transcription of the ribosomal genes is coupled to cell cycle arrest induced by cell density. Interestingly with an increase in cell density (from 3 - 43% confluence), a significant accumulation in the cellular content of hyperphosphorylated Rb was observed. As cell density increased further, the hypophosphorylated form of Rb became predominant and accumulated in the nucleoli. Co-immunoprecipitation experiments demonstrated there was also a significant rise in the amount of hypophosphorylated Rb associated with the rDNA transcription factor UBF. This increased interaction between Rb and UBF correlated with the reduced rate of rDNA transcription. Furthermore, overexpression of recombinant Rb inhibited UBF-dependent activation of transcription from a cotransfected rDNA reporter in either confluent or exponential cells. The amounts or activities of the rDNA transcription components we examined did not significantly change with cell cycle arrest. Although the content of PAF53, a polymerase associated factor, was altered marginally (decreased 38%), the time course and magnitude of the decrease did not correlate with the reduced rate of rDNA transcription. The results presented support a model wherein regulation of the binding of UBF to Rb and, perhaps the cellular content of PAF53, are components of the mechanism through which cell cycle and rDNA transcription are linked. Oncogene (2000) 19, 3487 - 3497

Regions upstream from the core promoter of the rat ribosomal gene are required for the formation of a stable transcription initiation complex by RNA polymerase I in vitro.

The sites required for the formation of a stable transcription initiation complex and for the initiation of transcription of rat rDNA in vitro were examined. A series of 5' deletion mutants of the rat transcription initiation region (-167 through +638) were constructed. These mutants were examined for their ability to support the faithful initiation of transcription in vitro. Mutants which contain less than 31 nucleotides upstream of the initiation site (+1) were unable to support detectable initiation of transcription. In this transcription system a series of deletion mutants from -167 to -31 were transcribed with equal efficiency when assayed individually. On the other hand, when the wild-type and mutant templates were compared in order-of-addition assays, they were found to be unequal. The incubation of an extract with a wild-type template, prior to the addition of nucleotides, precluded transcription of any second template added after the preincubation step. However, the preincubation of extract with mutants of the region upstream of the core promoter, from -122 to -31, did not preclude transcription of a wild-type template added after the preincubation step. Formation of the stable preinitiation complex was found to require the region between and -167.

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.

Characterization of rat ribosomal DNA. The highly repetitive sequences that flank the ribosomal RNA transcription unit are homologous and contain RNA polymerase III transcription initiation sites.

The non-transcribed spacers (NTS) of the ribosomal genes of a number of organisms have been studied and were found to contain repetitive sequences. In these studies with plasmid subclones of NTS, designated p3.4, p2.6 and p1.7, which come from both 5' and 3' flanking regions of the rat ribosomal genes, respectively, it has been determined that these sequences are found elsewhere within the genome. Southern hybridization analysis has demonstrated that the 5' and 3' NTS subclones cross-hybridize, and that the cross-hybridizing regions are synonymous with the highly repetitive regions. Sequences homologous to the rat NTS were specifically localized to both 5' and 3' flanking regions as well as to a number of the introns of cloned genes including rat serum albumin, rat alpha-fetoprotein, rat casein and human serum albumin. No hybridization was detected of the 5' NTS subclone to the human Alu sequence clone, Blur 8, or to the rodent equivalent, a clone containing Chinese hamster ovary type I and II Alu sequences. However, as reported for type II Alu sequences, the subcloned rat NTS sequences contain RNA polymerase III initiation sites and also hybridize to a number of small RNAs, but not 4.5 S or 7 S RNA. Sequence analysis of two distinct repetitive regions in p1.7 has revealed a region of alternating purine-pyrimidine nucleotides, potentially of Z DNA, and stretches of repetitive sequences. The possible roles for these repetitive sequences in recombination and in maintaining a hierarchical structure for the ribosomal genes are discussed.

Characterization of rat ribosomal DNA II. identification of the highly repetitive DNA in the 3' non-transcribed spacer.

The nucleotide sequence of one of the non-transcribed spacer subclones, p1.7, from the region 3' to rat 45 S pre-rRNA has been determined. Within 1612 base-pairs, the fragment contains two distinct regions of highly repetitive DNA, one of which can serve as a site for initiation in vitro by RNA polymerase III. The first is the alternating purine-pyrimidine sequence (A-C)21. The second of these regions has 95% homology to the identifier sequence and served as the template for RNA polymerase III transcription in vitro. The in vitro polymerase III template is aligned in opposite polarity to the direction of transcription of 45 S rRNA. Located near the identifier sequence is a region that is 59% homologous to the type-II Alu sequences. It would seem, therefore, that members of more than one highly repetitive sequence family have accumulated in the non-transcribed spacers. These data also suggest that within the non-transcribed spacers these families have evolved (sequence variation) at different rates, until one of them, the Alu type-II-like element, may represent a new Alu type-II subfamily.

rDNA transcription and cardiac hypertrophy.

A common result of the various stimuli that alter the growth rates of cells is a parallel alteration of the rate of ribosome biogenesis. In many cell types, this mandates an altered rate of ribosomal RNA synthesis. Preliminary studies indicate that rDNA transcription may in fact be regulated at more than one level or step. Our understanding of the process of transcription and the mechanisms by which it is regulated are discussed.

Regulation of ribosomal DNA transcription during neonatal cardiomyocyte hypertrophy.

In summary, the increased capacity for protein synthesis that is a constant feature in all forms of cardiac hypertrophy is largely mediated by accelerated ribosome biogenesis. Experiments with neonatal cardiomyocytes in culture indicate that the activity of the rDNA transcription factor, UBF, may contribute to the regulation of rDNA transcription during the hypertrophic growth process. The observations of parallel responses in three different models of neonatal cardiomyocyte hypertrophy suggest that further studies on the regulation of UBF should lead to a clearer understanding of the pathways that lead to hypertrophy. Possible alterations in the activities and/or amounts of other factors associated with rDNA transcription including SL-1, TFIC and the polymerase I enzyme itself, may also contribute to the regulation of cardiomyocyte growth; however, this remains to be demonstrated.

Isolation and characterization of rat ribosomal DNA clones.

Four EcoRI fragments, which contain the transcribed portion of the rat rDNA repeat, have been isolated from a rat genome library cloned in lambda Charon 4A vector. Three of the fragments, 9.6, 6.7, and 4.5 kb, from clones lambda ChR-B4, lambda Nr-42, and lambda ChR-C4B9, contained part of the 5'-NTS, the 5'-ETS, 18S rDNA, ITS-1, 5.8S rDNA, 28S rDNA and approximately 3.5 kb of the 3'-NTS. Two EcoRI fragments, from clones lambda ChR-B4 and lambda ChR-B7E12, which coded for the 5'-NTS, the ETS, and most of the 18S rDNA, differed by 1 kb near the EcoRI site upstream of the 5' terminus of 18S rRNA. Restriction maps of the cloned DNA fragments were constructed by cleavage of the fragments with various restriction endonucleases and Southern hybridization with 18S, 5.8S, and 28S rRNA. These maps were confirmed and extended by subcloning several regions of the repeat in pBR322.