The circadian clock coordinates ribosome biogenesis.

Biological rhythms play a fundamental role in the physiology and behavior of most living organisms. Rhythmic circadian expression of clock-controlled genes is orchestrated by a molecular clock that relies on interconnected negative feedback loops of transcription regulators. Here we show that the circadian clock exerts its function also through the regulation of mRNA translation. Namely, the circadian clock influences the temporal translation of a subset of mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. Moreover, the circadian oscillator directly regulates the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis.

The HBx oncoprotein of hepatitis B virus potentiates cell transformation by inducing c-Myc-dependent expression of the RNA polymerase I transcription factor UBF.

BACKGROUND: The HBx oncoprotein of hepatitis B virus has been implicated in the development and progression of hepatocellular carcinoma (HCC). HBx engages multiple signalling and growth-promoting pathways to induce cell proliferation and enhance ribosome biogenesis. Interestingly, the levels of Upstream Binding Factor (UBF) required for rDNA transcription and ribosome biogenesis are found elevated in the HCC patients. However, the molecular mechanism of UBF overexpression under the HBx microenvironment and consequent cell transformation remains elusive. METHODS: The UBF gene expression was investigated after co-expressing HBx in immortalized human hepatocytes (IHH) and human hepatoma Huh7 cells. Gene expression analysis involved estimation of mRNA level by real-time PCR, western blotting of protein, chromatin immune-precipitation assay, BrdU incorporation assay and soft agar colony formation assay. UBF expression was also investigated in an HBx transgenic mouse model of HCC to get a better mechanistic insight under more physiological conditions. RESULTS: Ectopic expression of HBx in IHH as well as Huh7 cells led to a marked increase in UBF expression both at mRNA and protein levels. Elevated levels of UBF were also observed in the hepatic tumors of HBx transgenic mice. Our ChIP studies revealed a marked increase in the occupancy of c-Myc on the UBF gene promoter in the presence of HBx and increase in its transcription. Enhanced UBF expression under the HBx microenvironment led to a marked increase in cell proliferation and transformation of IHH cells. CONCLUSIONS: Our study provides some compelling evidences in support of HBx-mediated increase in UBF levels that abets oncogenic onslaught in hepatic cells by increasing rDNA transcription and ribosome biogenesis.

c-MYC coordinately regulates ribosomal gene chromatin remodeling and Pol I availability during granulocyte differentiation.

Loss of c-MYC is required for downregulation of ribosomal RNA (rRNA) gene (rDNA) transcription by RNA Polymerase I (Pol I) during granulocyte differentiation. Here, we demonstrate a robust reduction of Pol I loading onto rDNA that along with a depletion of the MYC target gene upstream binding factor (UBF) and a switch from epigenetically active to silent rDNA accompanies this MYC reduction. We hypothesized that MYC may coordinate these mechanisms via direct regulation of multiple components of the Pol I transcription apparatus. Using gene expression arrays we identified a 'regulon' of Pol I factors that are both downregulated during differentiation and reinduced in differentiated granulocytes upon activation of the MYC-ER transgene. This regulon includes the novel c-MYC target genes RRN3 and POLR1B. Although enforced MYC expression during granulocyte differentiation was sufficient to increase the number of active rRNA genes, its activation in terminally differentiated cells did not alter the active to inactive gene ratio despite increased rDNA transcription. Thus, c-MYC dynamically controls rDNA transcription during granulocytic differentiation through the orchestrated transcriptional regulation of core Pol I factors and epigenetic modulation of number of active rRNA genes.

Changes in caveolae, caveolin, and polymerase 1 and transcript release factor (PTRF) expression in prostate cancer progression.

BACKGROUND: Caveolae are specialized invaginations in the cell membrane involved in the regulation of cell transport and signal transduction. The aims of this study were to investigate the number of caveolae and expression of caveolae-associated proteins, caveolin-1 and -2, and polymerase 1 and transcript release factor (PTRF) with development of prostate cancer. METHODS: Transmission electron microscopy was used to investigate the number of caveolae in normal human prostate stromal, epithelial cells, and androgen-dependent (LNCaP) and androgen-independent (PC3) cancer cell lines. Surgical tissue was obtained from patients with benign prostatic hyperplasia (BPH), well-differentiated and poorly differentiated prostate cancer. Caveolin-1, caveolin-2, and PTRF expression was identified by immunohistochemistry in tissue samples and quantified by Western blot analysis in cell lines. RESULTS: Caveolae were identified in normal epithelial and stromal prostate cells. The number of caveolae was significantly reduced in LNCaP and PC3 cells (P < 0.0001). PTRF was localized to stromal and epithelial cells in tissue from patients with BPH and in normal stromal and epithelial cells in vitro. PTRF expression was significantly decreased in LNCaP and PC3 cells and also in cancer tissue. In prostate tissue, caveolin-1 and -2 expression appeared to increase in prostate cancer. Caveolin-1 and -2 expression was decreased in LNCaP cells but caveolin-2 expression was significantly increased in PC3 cells compared to normal epithelial cells. CONCLUSIONS: This study demonstrates that changes in the cell membrane involving loss of caveolae and PTRF expression occur with the development of prostate cancer. These changes are accompanied by an up-regulation of caveolin-2.

TNFAIP8 regulates cisplatin resistance through TAF-Iα and promotes malignant progression of esophageal cancer.

OBJECTIVE: Previous studies have demonstrated that TNFAIP8 is a cancer-promoting gene. However, the role of TNFAIP8 in esophageal cancer (ECa) has not been reported. The aim of this investigation was to investigate the expression of TNFAIP8 in ECa, and to further explore whether it could regulate cisplatin resistance to this cancer via modulating TAF-Iα expression and promote malignant progression of ECa. PATIENTS AND METHODS: Quantitative Real Time-PCR (qRT-PCR) was performed to examine the expression of TNFAIP8 in 47 tumor tissue specimens and adjacent ones of ECa patients, and the interplay between TNFAIP8 expression and prognosis of patients with ECa was then analyzed. Further, qRT-PCR was applied to verify TNFAIP8 level in ECa cell lines. In addition, the TNFAIP8 knockdown model was constructed in ECa cisplatin-resistant cell lines including EC-109/DDP and OE19/DDP, and then, CCK8 and transwell assays were performed to analyze the impact of TNFAIP8 on the biological function of ECa cells; meanwhile, the Luciferase reporter gene assay and cell reverse experiments were finally conducted to explore its underlying mechanisms. RESULTS: The qRT-PCR results revealed that the TNFAIP8 level in tumor tissue samples of ECa patients was remarkably higher than that in adjacent ones, and the difference was statistically significant. Similarly, the overall survival rate of patients with high expression of TNFAIP8 was lower when compared with patients with low expression of TNFAIP8. EC-109/DDP and OE19/DDP, the ECa cisplatin-resistant cell lines, were successfully constructed; subsequently, it was found that the proliferation, invasiveness, and metastasis ability of ECa cells in TNFAIP8 knockdown group was remarkably decreased compared with those in the sh-NC group. At the same time, the Western blot results illustrated that the expression of TAF-Iαwas remarkably elevated in the TNFAIP8 knockdown group. In addition, the Luciferase reporting assay and cell reverse experiments also demonstrated that there existed a mutual regulation effect between TNFAIP8 and TAF-Iα, which might together affect the malignant progression of ECa. CONCLUSIONS: The expression of TNFAIP8 was found remarkably enhanced in ECa tissues and cell lines, which might be closely relevant to the poor prognosis of patients with ECa. Additionally, it was found that TNFAIP8 may regulate cisplatin resistance and promote malignant progression of ECa by modulating TAF-Iα expression.

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.

[Activation of transcription of ribosome genes following human embryo fibroblast infection with cytomegalovirus in vitro]. / Aktivatsiia transkriptsii ribosomnykh genov pri tsitomegalovirusnoi infektsii fibroblastov émbriona cheloveka in vitro.

Cytomegalovirus (CMV) infection of human diploid embryo fibroblasts in vitro causes a massive cell death on the 3-4th day of infection with a high primary infection coefficient (1-5 U/cell). Cytopathological effects of viral infection on the 3-4th day includes diminishes of the cell size, changes in their form, compaction of the nuclear chromatin, and disorganization and inactivation of the nucleolus. However, the early stages of the viral infection progression (the 1st-2nd day) are accompanied by unequivocal activation of rDNA (ribosomal gene) and the bulk of chromatin transcription. There are several features to support this conclusion: In the early CMV-infected cell 1) the nucleolar size is increased; 2) the number of intranucleolar foci binding the specific RNA-polymerase I transcription factor (UBF) is augmented; 3) the Ag-NOR staining is enhanced; 4) 3H-uridine incorporation to the nucleoli is activated; 5) the ultrastructure of the nucleolus is changed. Altogether, these data argue in favor of activation of rDNA transcription in human fibroblasts in vitro at the initial stages of infection.

Nuclear and nucleolar reprogramming during the first cell cycle in bovine nuclear transfer embryos.

The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed at 0.5, 1, 2, 3, 4, 8, 12, and 16 h postactivation (hpa). Parthenogenetic (PA) embryos were used as control. The SCNT and PA embryos were processed for lacmoid staining, autoradiography, transmission electron microscopy (TEM), and immunofluorescence localization of: upstream binding factor (UBF) and fibrillarin at 4 and 12 hpa. Likewise, starved and nonstarved fibroblasts were processed for autoradiography and TEM. The fibroblasts displayed strong transcriptional activity and active fibrillogranular nucleoli. None of the reconstructed embryos, however, displayed transcriptional activity. In conclusion, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development.

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

rRNA transcription is a fundamental requirement for all cellular growth processes and is activated by the phosphorylation of the upstream binding factor (UBF) in response to growth stimulation. Even though it is well known that phosphorylation of UBF is required for its activation and is a key step in activation of rRNA transcription, as yet, there has been no direct mapping of the UBF phosphorylation sites. The results of the present studies employed sophisticated nano-flow HPLC-microelectrospray-ionization tandem mass spectrometry (nHPLC-muESI-MS/MS) coupled with immobilized metal affinity chromatography (IMAC) and computer database searching algorithms to identify 10 phosphorylation sites on UBF at serines 273, 336, 364, 389, 412, 433, 484, 546, 584, and 638. We then carried out functional analysis of two of these sites, serines 389 and 584. Serine-alanine substitution mutations of 389 (S389A) abrogated rRNA transcription in vitro and in vivo, whereas mutation of serine 584 (S584A) reduced transcription in vivo but not in vitro. In contrast, serine-glutamate mutation of 389 (S389E) restored transcriptional activity. Moreover, S389A abolished UBF-SL1 interaction in vitro, while S389E partially restored UBF-SL1 interaction. Taken together, the results of these studies suggest that growth factor stimulation induces an increase in rRNA transcriptional activity via phosphorylation of UBF at serine 389 in part by facilitating a rate-limiting step in the recruitment of RNA polymerase I: i.e., recruitment of SL1. Moreover, studies provide critical new data regarding multiple additional UBF phosphorylation sites that will require further characterization by the field.

Nucleolar protein upstream binding factor is sequestered into adenovirus DNA replication centres during infection without affecting RNA polymerase I location or ablating rRNA synthesis.

When human adenovirus infects human cells there is disruption of rRNA biogenesis. This report examines the effect of adenovirus infection on the nucleolar protein, upstream binding factor (UBF) which plays a major role in regulating rRNA synthesis. We determined that early after infection, UBF associates with the replication of viral DNA, preferentially associating with the ends of the linear viral genome, and that addition of anti-UBF serum to in vitro replication assays markedly reduced viral DNA replication. Regions of UBF important to these observations are also established. Interestingly, sequestering the majority of UBF from the nucleolus did not lead to the ablation of rRNA synthesis or the sequestration of RNA pol I. In infected cells the bulk of RNA synthesis was RNA pol I associated and distinct from the location of most of the detectable UBF. We propose that UBF plays a role in viral DNA replication, further strengthening the role of nucleolar antigens in the adenovirus life cycle.

Regulation of upstream binding factor 1 activity by insulin-like growth factor I receptor signaling.

The upstream binding factor 1 (UBF1) is one of the proteins in a complex that regulates the activity of RNA polymerase I, which controls the rate of ribosomal RNA (rRNA) synthesis. We have shown previously that insulin receptor substrate-1 (IRS-1) can translocate to the nuclei and nucleoli of cells and bind UBF1. We report here that activation of the type I insulin-like growth factor receptor (IGF-IR) by IGF-I increases transcription from the ribosomal DNA (rDNA) promoter in both myeloid cells and mouse fibroblasts. The increased activity of the rDNA promoter is accompanied by increased phosphorylation of UBF1, a requirement for UBF1 activation. Phosphorylation occurs on a number of UBF1 peptides, most prominently on the highly acidic, serine-rich C terminus. In myeloid cells (but not in mouse embryo fibroblasts) IRS-1 signaling stabilizes the levels of UBF1 protein. These findings demonstrate that IGF-IR signaling can increase the activity of UBF1 and transcription from the rDNA promoter, providing one explanation for the reported effects of the IGF/IRS-1 axis on cell and body size in animals and cells in culture.