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Mycophenolic acid (MPA) is the active metabolite of mycophenolate mofetil, a drug that is widely used for immunosuppression in organ transplantation and autoimmune diseases, as well as anticancer chemotherapy. It inhibits IMP dehydrogenase, a rate-limiting enzyme in de novo synthesis of guanidine nucleotides. MPA treatment interferes with transcription elongation, resulting in a drastic reduction of pre-rRNA and pre-tRNA synthesis, the disruption of the nucleolus, and consequently cell cycle arrest. Here, we investigated the mechanism whereby MPA inhibits RNA polymerase III (Pol III) activity, in both yeast and mammalian cells. We show that MPA rapidly inhibits Pol III by depleting GTP. Although MPA treatment can activate p53, this is not required for Pol III transcriptional inhibition. The Pol III repressor MAF1 is also not responsible for inhibiting Pol III in response to MPA treatment. We show that upon MPA treatment, the levels of selected Pol III subunits decrease, but this is secondary to transcriptional inhibition. Chromatin immunoprecipitation (ChIP) experiments show that Pol III does not fully dissociate from tRNA genes in yeast treated with MPA, even though there is a sharp decrease in the levels of newly transcribed tRNAs. We propose that in yeast, GTP depletion may lead to Pol III stalling., (Copyright © 2019 Jurkiewicz et al.)

A million copies of the Alu short interspersed nuclear element (SINE) are scattered throughout the human genome, providing ∼11% of our total DNA. SINEs spread by retrotransposition, using a transcript generated by RNA polymerase (pol) III from an internal promoter. Levels of these pol III-dependent Alu transcripts are far lower than might be expected from the abundance of the template. This was believed to reflect transcriptional suppression through DNA methylation, denying pol III access to most SINEs through chromatin-mediated effects. Contrary to expectations, our recent study found no evidence that methylation of SINE DNA reduces its occupancy or expression by pol III. However, histone H3 associated with SINEs is prominently methylated on lysine 9, a mark that correlates with transcriptional silencing. The SUV39 methyltransferases that deposit this mark can be found at many SINEs. Furthermore, a selective inhibitor of SUV39 stimulates pol III recruitment to these loci, as well as SINE expression. These data suggest that methylation of histone H3 rather than DNA may mediate repression of SINE transcription by pol III, at least under the conditions we studied.

Inflammation in the tumor microenvironment has many tumor-promoting effects. In particular, tumor-associated macrophages (TAMs) produce many cytokines which can support tumor growth by promoting survival of malignant cells, angiogenesis, and metastasis. Enhanced cytokine production by TAMs is tightly coupled with protein synthesis. In turn, translation of proteins depends on tRNAs, short abundant transcripts that are made by RNA polymerase III (Pol III). Here, we connect these facts by showing that stimulation of mouse macrophages with lipopolysaccharides (LPS) from the bacterial cell wall causes transcriptional upregulation of tRNA genes. The transcription factor NF-κB is a key transcription factor mediating inflammatory signals, and we report that LPS treatment causes an increased association of the NF-κB subunit p65 with tRNA genes. In addition, we show that p65 can directly associate with the Pol III transcription factor TFIIIB and that overexpression of p65 induces Pol III-dependent transcription. As a consequence of these effects, we show that inhibition of Pol III activity in macrophages restrains cytokine secretion and suppresses phagocytosis, two key functional characteristics of these cells. These findings therefore identify a radical new function for Pol III in the regulation of macrophage function which may be important for the immune responses associated with both normal and malignant cells., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Detailed hourly precipitation data are required for long-range modeling of dispersion and wet deposition of particulate matter and water-soluble pollutants using the CALPUFF model. In sparsely populated areas such as the north central United States, ground-based precipitation measurement stations may be too widely spaced to offer a complete and accurate spatial representation of hourly precipitation within a modeling domain. The availability of remotely sensed precipitation data by satellite and the National Weather Service array of next-generation radars (NEXRAD) deployed nationally provide an opportunity to improve on the paucity of data for these areas. Before adopting a new method of precipitation estimation in a modeling protocol, it should be compared with the ground-based precipitation measurements, which are currently relied upon for modeling purposes. This paper presents a statistical comparison between hourly precipitation measurements for the years 2006 through 2008 at 25 ground-based stations in the north central United States and radar-based precipitation measurements available from the National Center for Environmental Predictions (NCEP) as Stage IV data at the nearest grid cell to each selected precipitation station. It was found that the statistical agreement between the two methods depends strongly on whether the ground-based hourly precipitation is measured to within 0.1 in/ hr or to within 0.01 in/hr. The results of the statistical comparison indicate that it would be more accurate to use gridded Stage IV precipitation data in a gridded dispersion model for a long-range simulation, than to rely on precipitation data interpolated between widely scattered rain gauges., Implications: The current reliance on ground-based rain gauges for precipitation events and hourly data for modeling of dispersion and wet deposition of particulate matter and water-soluble pollutants results in potentially large discontinuity in data coverage and the need to extrapolate data between monitoring stations. The use of radar-based precipitation data, which is available for the entire continental United States and nearby areas, would resolve these data gaps and provide a complete and accurate spatial representation of hourly precipitation within a large modeling domain.

It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.

RNA polymerase (pol) III manufactures transfer RNAs, 5S ribosomal RNA and several other untranslated RNA molecules that are essential components of the biosynthetic process. Accordingly, transcription by pol III is closely coupled to cellular growth rate. In mammals, stringent regulation of pol III output is achieved through the concerted action of various mechanisms that target the essential pol III-specific transcription factor TFIIIB. Positive regulators of growth, including ERK and c-Myc, directly bind and activate TFIIIB, thus increasing pol III output when growth demands are high. In contrast, TFIIIB is inactive when bound by RB. Growth stimulation leads to RB hyperphosphorylation, which alleviates this repression. These TFIIIB-directed mechanisms regulate pol III transcription in proliferating fibroblasts, and this is likely to contribute to the tight coordination of cell growth with division. Recent evidence indicates that these same pol III-regulatory mechanisms operate in terminally differentiated cells, where growth occurs in the absence of division, leading to hypertrophic enlargement. This cell division-independent regulation of pol III transcription, and hence biosynthetic capacity, is consistent with a direct involvement of these proteins in controlling cell growth. ERK-mediated induction of expression of the TFIIIB subunit Brf1 was identified as an additional mechanism for raising pol III output in terminally differentiated cardiomyocytes. Brf1 levels are limiting for pol III transcription in resting cardiomyocytes and so hypertrophic stimuli induce Brf1 expression as part of the pol III response in this context. The complex network of strategies that couple pol III transcription with cell growth suggest that stringent control of this system is of fundamental importance.

ProgeniDB is a web-accessible cell lineage database, which comprises data derived from time-lapse microscopy image sequences, using our novel encoding software tool called ProgeniTRAK. This database provides information on qualitative and quantitative changes in cellular dynamics in response to anti-cancer drugs variation, which is critical for pharmacodynamic modeling and validation. The lineage data for each progenitor cell centers around the construction of a bifurcation map, locating in both space and time critical cellular events such as cell division and cell death. This database provides the opportunity to select a set of lineages based on different experimental constraints and observe the associated phenotypic behavior of progenitor cells. The output generated from this database depicts the cellular dynamic of selected population which can be downloaded to explore and exploit in relation to hypothesis-driven drug discovery. A case study is presented where we interrogate drug-induced perturbations on ensuing cell lineages. The database is available at http://biodiversity.cs.cf.ac.uk/cymart/progenidb.html.

The TATA-binding protein, TBP, is used by all three RNA polymerases and is therefore central to the process of gene expression. TBP associates with several subsets of proteins, called TATA-binding protein-associated factors (TAFs). This results in the formation of at least three distinct complexes, SL1, TFIID, and TFIIIB, which dictates whether TBP functions in RNA polymerase (pol) I, pol II, or pol III transcription, respectively. The regulation of gene expression has focused largely on proteins that serve to modulate the efficiency by which the general transcription components, such as TBP, interact with promoters. The possibility of a basal transcription factor, itself, being regulated, and influencing cellular homeostasis, has not been extensively considered. However, recent studies have indicated that TBP is indeed regulated, and that modulation of its cellular concentration has a profound, and surprisingly selective, impact on gene expression that can mediate the normal proliferative responses of cells to growth stimuli as well as the transformation potential of cells.

The synthesis of tRNA and 5S rRNA by RNA polymerase (pol) III is cell cycle regulated in higher organisms. Overexpression of pol III products is a general feature of transformed cells. These observations may be explained by the fact that a pol III-specific transcription factor, TFIIIB, is strongly regulated by the tumor suppressors RB and p53, as well as the proto-oncogene product c-Myc. RB and p53 repress TFIIIB, but this restraint can be lost in tumors through a variety of mechanisms. In contrast, c-Myc binds and activates TFIIIB, causing potent induction of pol III transcription. Using chromatin immunoprecipitation and RNA interference, we show that c-Myc interacts with tRNA and 5S rRNA genes in transformed cervical cells, stimulating their expression. Availability of pol III products may be an important determinant of a cell's capacity to grow. The ability to regulate pol III output may therefore be integral to the growth control functions of RB, p53 and c-Myc.

CK2 is a highly conserved protein kinase with growth-promoting and oncogenic properties. It is known to activate RNA polymerase III (PolIII) transcription in Saccharomyces cerevisiae and is shown here to also exert a potent effect on PolIII in mammalian cells. Peptide and chemical inhibitors of CK2 block PolIII transcription in human cell extracts. Furthermore, PolIII transcription in mammalian fibroblasts is decreased significantly when CK2 activity is compromised by chemical inhibitors, antisense oligonucleotides, or kinase-inactive mutants. Coimmunoprecipitation and cofractionation show that endogenous human CK2 associates stably and specifically with the TATA-binding protein-containing factor TFIIIB, which brings PolIII to the initiation site of all class III genes. Serum stimulates TFIIIB phosphorylation in vivo, an effect that is diminished by inhibitors of CK2. Binding to TFIIIC2 recruits TFIIIB to most PolIII promoters; this interaction is compromised specifically by CK2 inhibitors. The data suggest that CK2 stimulates PolIII transcription by binding and phosphorylating TFIIIB and facilitating its recruitment by TFIIIC2. CK2 also activates PolI transcription in mammals and may therefore provide a mechanism to coregulate the output of PolI and PolIII. CK2 provides a rare example of an endogenous activity that operates on the PolIII system in both mammals and yeasts. Such evolutionary conservation suggests that this control may be of fundamental importance.

The "pocket" proteins pRb, p107, and p130 are a family of negative growth regulators. Previous studies have demonstrated that overexpression of pRb can repress transcription by RNA polymerase (Pol) I. To assess whether pRb performs this role under physiological conditions, we have examined pre-rRNA levels in cells from mice lacking either pRb alone or combinations of the three pocket proteins. Pol I transcription was unaffected in pRb-knockout fibroblasts, but specific disruption of the entire pRb family deregulated rRNA synthesis. Further analysis showed that p130 shares with pRb the ability to repress Pol I transcription, whereas p107 is ineffective in this system. Production of rRNA is abnormally elevated in Rb(-/-) p130(-/-) fibroblasts. Furthermore, overexpression of p130 can inhibit an rRNA promoter both in vitro and in vivo. This reflects an ability of p130 to bind and inactivate the upstream binding factor, UBF. The data imply that rRNA synthesis in living cells is subject to redundant control by endogenous pRb and p130.

The retinoblastoma protein (RB) has been shown to suppress RNA polymerase (Pol) III transcription in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88-90, 1996). This regulation involves interaction with TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061-2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755-14761, 1997). However, it has not been established why RB binding to TFIIIB results in transcriptional repression. For several Pol II-transcribed genes, RB has been shown to inhibit expression by recruiting histone deacetylases, which are thought to decrease promoter accessibility. We present evidence that histone deacetylases exert a negative effect on Pol III activity in vivo. However, RB remains able to regulate Pol III transcription in the presence of the histone deacetylase inhibitor trichostatin A. Instead, RB represses by disrupting interactions between TFIIIB and other components of the basal Pol III transcription apparatus. Recruitment of TFIIIB to most class III genes requires its binding to TFIIIC2, but this can be blocked by RB. In addition, RB disrupts the interaction between TFIIIB and Pol III that is essential for transcription. The ability of RB to inhibit these key interactions can explain its action as a potent repressor of class III gene expression.

RNA polymerase (Pol) III transcription is abnormally active in fibroblasts that have been transformed by simian virus 40 (SV40). This report presents evidence that two separate components of the general Pol III transcription apparatus, TFIIIB and TFIIIC2, are deregulated following SV40 transformation. TFIIIC2 subunits are expressed at abnormally high levels in SV40-transformed cells, an effect which is observed at both protein and mRNA levels. In untransformed fibroblasts, TFIIIB is subject to repression through association with the retinoblastoma protein RB. The interaction between RB and TFIIIB is compromised following SV40 transformation. Furthermore, the large T antigen of SV40 is shown to relieve repression by RB. The E7 oncoprotein of human papillomavirus can also activate Pol III transcription, an effect that is dependent on its ability to bind to RB. The data provide evidence that both TFIIIB and TFIIIC2 are targets for activation by DNA tumor viruses.

RNA polymerase III (Pol III) transcription is subject to repression by the retinoblastoma protein RB, both in vitro and in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88-90, 1996). This is achieved through a direct interaction between RB and TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061-2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755-14761, 1997). p107 and p130 are two closely related proteins that display 30 to 35% identity with the RB polypeptide and share some of its functions. We show that p107 and p130 can both repress Pol III transcription in transient transfection assays or when added to cell extracts. Pull-down assays and immunoprecipitations using recombinant components demonstrate that a subunit of TFIIIB interacts physically with p107 and p130. In addition, endogenous TFIIIB is shown by cofractionation and coimmunoprecipitation to associate stably with both p107 and p130. Disruption of this interaction in vivo by using the E7 oncoprotein of human papillomavirus results in a marked increase in Pol III transcription. Pol III activity is also deregulated in fibroblasts derived from p107 p130 double knockout mice. We conclude that TFIIIB is targeted for repression not only by RB but also by its relatives p107 and p130.

We have identified a putative gene within the Caenorhabditis elegans genome which has the potential to encode a protein homologous to BRF, an RNA polymerase III general transcription factor. The predicted protein shares very similar overall structure with human and yeast BRF. In particular, its N-terminal half comprises a zinc-ribbon motif and a TR domain which is also present in the cyclin box. The C. elegans protein is more similar to human BRF than to the yeast BRF proteins, as would be expected from an evolutionary standpoint. Alignment of the C. elegans protein with the four known BRF proteins reveals two blocks conserved between all five sequences within the diverged C-terminal region. Profile searches using these regions suggest that they may contain evolutionarily conserved motifs. These comparisons provide insight into the structure and function of an important transcription factor.

Following an historical review of earlier attempts to develop separated head and brain preparations and their contributions to modern-day understanding of the neurophysiology and neurochemistry of the central nervous system, the experiments that eventually led to the first successful total isolation of the mammalian brain are presented. The operative strategies emphasizing the anatomical and physiological problems requiring solution that resulted in vascular and neurogenic separation from the parent body and cephalon are described. The innovative engineering concepts that were utilized in the design of miniaturized equipment to maintain the isolated brain in a living state under conditions of cross circulation, extracorporeal artificial perfusion and transplantation are elaborated. Investigations employing isolated brain and cephalic preparation documenting tissue substrate requirements, metabolic and rheological conditions prevailing at various low temperatures and the immunologically privileged state of the separated organ are briefly presented. The unique opportunities these isolated brain models offer for study are emphasized as well as the complexity of their surgical preparation, which, to date, has limited their universal applications.

Inactivation of the TATA-binding protein-containing complex TFIIIB contributes to the mitotic repression of RNA polymerase III transcription, both in frogs and in humans (J. M. Gottesfeld, V. J. Wolf, T. Dang, D. J. Forbes, and P. Hartl, Science 263:81-84, 1994; R. J. White, T. M. Gottlieb, C. S. Downes, and S. P. Jackson, Mol. Cell. Biol. 15:1983-1992, 1995). Using extracts of synchronized proliferating HeLa cells, we show that TFIIIB activity remains low during the early part of G1 phase and increases only gradually as cells approach S phase. As a result, the transcription of all class III genes tested is significantly less active in early G1 than it is in S or G2 phase, both in vitro and in vivo. The increased activity of TFIIIB as cells progress through interphase appears to be due to changes in the TATA-binding protein-associated components of this complex. The data suggest that TFIIIB is an important target for the cell cycle regulation of RNA polymerase III transcription during both mitosis and interphase of actively proliferating HeLa cells.

The mitotic state is associated with a generalized repression of transcription. We show that mitotic repression of RNA polymerase III transcription can be reproduced by using extracts of synchronized HeLa cells. We have used this system to investigate the molecular basis of transcriptional repression during mitosis. We find a specific decrease in the activity of the TATA-binding-protein (TBP)-containing complex TFIIIB. TBP itself is hyperphosphorylated at mitosis, but this does not appear to account for the loss of TFIIIB activity. Instead, one or more TBP-associated components appear to be regulated. The data suggest that changes in the activity of TBP-associated components contribute to the coordinate repression of gene expression that occurs at mitosis.