Basic mechanisms in RNA polymerase I transcription of the ribosomal RNA genes.

RNA Polymerase (Pol) I produces ribosomal (r)RNA, an essential component of the cellular protein synthetic machinery that drives cell growth, underlying many fundamental cellular processes. Extensive research into the mechanisms governing transcription by Pol I has revealed an intricate set of control mechanisms impinging upon rRNA production. Pol I-specific transcription factors guide Pol I to the rDNA promoter and contribute to multiple rounds of transcription initiation, promoter escape, elongation and termination. In addition, many accessory factors are now known to assist at each stage of this transcription cycle, some of which allow the integration of transcriptional activity with metabolic demands. The organisation and accessibility of rDNA chromatin also impinge upon Pol I output, and complex mechanisms ensure the appropriate maintenance of the epigenetic state of the nucleolar genome and its effective transcription by Pol I. The following review presents our current understanding of the components of the Pol I transcription machinery, their functions and regulation by associated factors, and the mechanisms operating to ensure the proper transcription of rDNA chromatin. The importance of such stringent control is demonstrated by the fact that deregulated Pol I transcription is a feature of cancer and other disorders characterised by abnormal translational capacity.

WT1 and its transcriptional cofactor BASP1 redirect the differentiation pathway of an established blood cell line.

The Wilms' tumour suppressor WT1 (Wilms' tumour 1) is a transcriptional regulator that plays a central role in organogenesis, and is mutated or aberrantly expressed in several childhood and adult malignancies. We previously identified BASP1 (brain acid-soluble protein 1) as a WT1 cofactor that suppresses the transcriptional activation function of WT1. In the present study we have analysed the dynamic between WT1 and BASP1 in the regulation of gene expression in myelogenous leukaemia K562 cells. Our findings reveal that BASP1 is a significant regulator of WT1 that is recruited to WT1-binding sites and suppresses WT1-mediated transcriptional activation at several WT1 target genes. We find that WT1 and BASP1 can divert the differentiation programme of K562 cells to a non-blood cell type following induction by the phorbol ester PMA. WT1 and BASP1 co-operate to induce the differentiation of K562 cells to a neuronal-like morphology that exhibits extensive arborization, and the expression of several genes involved in neurite outgrowth and synapse formation. Functional analysis revealed the relevance of the transcriptional reprogramming and morphological changes, in that the cells elicited a response to the neurotransmitter ATP. Taken together, the results of the present study reveal that WT1 and BASP1 can divert the lineage potential of an established blood cell line towards a cell with neuronal characteristics.

A cyclin D2-Rb pathway regulates cardiac myocyte size and RNA polymerase III after biomechanical stress in adult myocardium.

Normally, cell cycle progression is tightly coupled to the accumulation of cell mass; however, the mechanisms whereby proliferation and cell growth are linked are poorly understood. We have identified cyclin (Cyc)D2, a G(1) cyclin implicated in mediating S phase entry, as a potential regulator of hypertrophic growth in adult post mitotic myocardium. To examine the role of CycD2 and its downstream targets, we subjected CycD2-null mice to mechanical stress. Hypertrophic growth in response to transverse aortic constriction was attenuated in CycD2-null compared with wild-type mice. Blocking the increase in CycD2 in response to hypertrophic agonists prevented phosphorylation of CycD2-target Rb (retinoblastoma gene product) in vitro, and mice deficient for Rb had potentiated hypertrophic growth. Hypertrophic growth requires new protein synthesis and transcription of tRNA genes by RNA polymerase (pol) III, which increases with hypertrophic signals. This load-induced increase in RNA pol III activity is augmented in Rb-deficient hearts. Rb binds and represses Brf-1 and TATA box binding protein (TBP), subunits of RNA pol III-specific transcription factor B, in adult myocardium under basal conditions. However, this association is disrupted in response to transverse aortic constriction. RNA pol III activity is unchanged in CycD2(-/-) myocardium after transverse aortic constriction, and there is no dissociation of TBP from Rb. These investigations identify an essential role for the CycD2-Rb pathway as a governor of cardiac myocyte enlargement in response to biomechanical stress and, more fundamentally, as a regulator of the load-induced activation of RNA pol III.

Hypoxic stress suppresses RNA polymerase III recruitment and tRNA gene transcription in cardiomyocytes.

RNA polymerase (pol) III transcription decreases when primary cultures of rat neonatal cardiomyocytes are exposed to low oxygen tension. Previous studies in fibroblasts have shown that the pol III-specific transcription factor IIIB (TFIIIB) is bound and regulated by the proto-oncogene product c-Myc, the mitogen-activated protein kinase ERK and the retinoblastoma tumour suppressor protein, RB. The principal function of TFIIIB is to recruit pol III to its cognate gene template, an activity that is known to be inhibited by RB and stimulated by ERK. We demonstrate by chromatin immunoprecipitation (ChIP) that c-Myc also stimulates pol III recruitment by TFIIIB. However, hypoxic conditions cause TFIIIB dissociation from c-Myc and ERK, at the same time as increasing its interaction with RB. Consistent with this, ChIP assays indicate that the occupancy of tRNA genes by pol III is significantly reduced, whereas promoter binding by TFIIIB is undiminished. The data suggest that hypoxia can inhibit pol III transcription by altering the interactions between TFIIIB and its regulators and thus compromising its ability to recruit the polymerase. These effects are independent of cell cycle changes.

Activation by c-Myc of transcription by RNA polymerases I, II and III.

The proto-oncogene product c-Myc can induce cell growth and proliferation. It regulates a large number of RNA polymerase II-transcribed genes, many of which encode ribosomal proteins, translation factors and other components of the biosynthetic apparatus. We have found that c-Myc can also activate transcription by RNA polymerases I and III, thereby stimulating production of rRNA and tRNA. As such, c-Myc may possess the unprecedented capacity to induce expression of all ribosomal components. This may explain its potent ability to drive cell growth, which depends on the accumulation of ribosomes. The activation of RNA polymerase II transcription by c-Myc is often inefficient, but its induction of rRNA and tRNA genes can be very strong in comparison. We will describe what is known about the mechanisms used by c-Myc to activate transcription by RNA polymerases I and II.

Repression of transcription by WT1-BASP1 requires the myristoylation of BASP1 and the PIP2-dependent recruitment of histone deacetylase.

The Wilms' tumor 1 protein WT1 is a transcriptional regulator that is involved in cell growth and differentiation. The transcriptional corepressor BASP1 interacts with WT1 and converts WT1 from a transcriptional activator to a repressor. Here, we demonstrate that the N-terminal myristoylation of BASP1 is required in order to elicit transcriptional repression at WT1 target genes. We show that myristoylated BASP1 binds to nuclear PIP2, which leads to the recruitment of PIP2 to the promoter regions of WT1-dependent target genes. BASP1's myristoylation and association with PIP2 are required for the interaction of BASP1 with HDAC1, which mediates the recruitment of HDAC1 to the promoter and elicits transcriptional repression. Our findings uncover a role for myristoylation in transcription, as well as a critical function for PIP2 in gene-specific transcriptional repression through the recruitment of histone deacetylase.

Regulation of RNA polymerase III transcription by Maf1 in mammalian cells.

RNA polymerase (pol) III produces essential components of the biosynthetic machinery; therefore, its output is tightly coupled with the rate of cell growth and proliferation. In Saccharomyces cerevisiae, Maf1 is an essential mediator of pol III repression in response to starvation. We demonstrate that a Maf1 ortholog is also used to restrain pol III activity in mouse and human cells. Mammalian Maf1 represses pol III transcription in vitro and in transfected fibroblasts. Furthermore, genetic deletion of Maf1 elevates pol III transcript expression, thus confirming the role of endogenous Maf1 as an inhibitor of mammalian pol III output. Maf1 is detected at chromosomal pol III templates in rodent and human cells. It interacts with pol III as well as its associated initiation factor TFIIIB and is phosphorylated in a serum-sensitive manner in vivo. These aspects of Maf1 function have been conserved between yeast and mammals and are therefore likely to be of fundamental importance in controlling pol III transcriptional activity.

Regulation of RNA polymerase III transcription during mammalian cell growth.

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.

Regulation of RNA polymerase III transcription during hypertrophic growth.

The cell division-independent growth of terminally differentiated cardiomyocytes is commonly associated with cardiovascular disease. We demonstrate that it is accompanied by a substantial rise in transcription by RNA polymerase (pol) III, which produces essential components of the biosynthetic apparatus, including 5S rRNA and tRNAs. This increase in transcription is achieved by changes in both the activity and level of the essential pol III-specific transcription factor TFIIIB. Erk and c-Myc, which directly activate TFIIIB in proliferating fibroblasts, also induce pol III transcription in growing cardiomyocytes. Furthermore, hypertrophic stimulation increases expression of the essential TFIIIB subunit Brf1, an effect not seen when fibroblasts proliferate. Erk mediates this induction of Brf1 expression and therefore contributes in at least two ways to pol III transcriptional activation during hypertrophy. Increased production of tRNA and 5S rRNA will contribute to the enhanced translational capacity required to sustain hypertrophic growth.