A test of the model that RNA polymerase III transcription is regulated by selective induction of the 110 kDa subunit of TFIIIC.

TFIIIC is a RNA polymerase (pol) III-specific DNA-binding factor that is required for transcription of tRNA and 5S rRNA genes. Active human TFIIIC consists of five subunits. However, an inactive form has also been isolated that lacks one of the five subunits, called TFIIIC110. A model was proposed in which pol III transcription might be regulated by the specific induction of TFIIIC110, allowing formation of active TFIIIC from the inactive form. We have tested this model by transient transfection of HeLa and HEK293 cells with a vector expressing TFIIIC110. We have also made stably transfected HeLa cell lines that carry a doxycycline-inducible version of the cDNA for TFIIIC110. We show that the induced TFIIIC110 enters the nucleus, binds other TFIIIC subunits and is recruited to tRNA and 5S rRNA genes in vivo. However, little or no effect is seen on the expression of pol III transcripts. The data argue against the model that pol III transcription can be effectively modulated through the specific induction of TFIIIC110.

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.

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.