Regulation of a TATA-binding Protein-associated Factor during Cellular Differentiation

Dubeau

Journal of Biological Chemistry

2008

107

121

RNA polymerase (pol) III transcription, responsible for the synthesis of various stable RNAs, including 5 S rRNAs and tRNAs, is regulated by oncogenic proteins and tumor suppressors. Although it is well established that RNA pol III-dependent transcription is deregulated in transformed cells and malignant tumors, it has not been determined whether this represents a cause or consequence of these processes. We show that Rat1a fibroblasts undergoing oncogenic transformation by the TATAbinding protein or c-Myc display enhanced RNA pol III transcription. Decreased expression of the RNA pol III-specific transcription factor Brf1 prevented this increase in RNA pol III transcription. Although the overall proliferation rates of these cells remained unchanged, the ability of cells to grow in an anchorage-independent manner and form tumors in mice was markedly reduced. Although overexpression of Brf1 modestly stimulated RNA pol III transcription, expression of a phosphomimic, Brf1-T145D, more significantly induced transcription. However, these increases in transcription were not sufficient to promote cellular transformation. Together, these results demonstrate that enhanced RNA pol III transcription is essential for anchorage-independent growth and tumorigenesis and that these events can be uncoupled from effects on anchorage-dependent proliferation. RNA polymerase (pol)2 III synthesizes a variety of small untranslated RNAs, including tRNA, 5 S rRNA, U6 RNA, and 7SL RNA. RNA pol III products play essential roles in protein synthesis, RNA processing, and protein trafficking. rRNA synthesis by RNA pol I and pol III is a limiting step in ribosome accumulation (1), and the overall rate of translation is determined by the number of ribosomes (2). Thus, the rate of RNA pol I and pol III transcription dictates the biosynthetic capacity of cells and may be an important determinant of a cell's capacity to grow. Consistent with this idea, decreasing RNA pol I transcription has been shown to result in reduced cellular growth rates (3). Recent results showed that decreased RNA pol III transcription by the bacterial compound Tagetin prevented hypertropic enlargement of cardiomyocytes (4). Activation of RNA pol III-dependent transcription may thus be needed for increased cell mass.RNA pol III transcription is regulated by a variety of tumor suppressors and oncogenes. Oncogenic Ras (5), c-Myc (6), and activated phosphatidylinositol 3-kinase (7) all serve to induce transcription, whereas the tumor suppressors Rb (8), p53 (9), and PTEN (7) repress RNA pol III transcription. The RNA pol III-specific transcription factor (TF) IIIB complex, composed of TATA-binding protein (TBP), Brf1, and Bdp1, is the target of these regulatory proteins. Both Rb and p53 interact directly with components of the TFIIIB complex to inhibit its function, whereas c-Myc stimulates RNA pol III transcription by directly associating with the TFIIIB complex and enhancing its recruitment to promoters. In contrast, through its ability to regulate phosphatidylinositol 3-ki...

Section: Methodsmentioning

confidence: 99%

Enhanced RNA Polymerase III-dependent Transcription Is Required for Oncogenic Transformation*

Dubeau

Journal of Biological Chemistry

2008

107

121

Molecular and Cellular Biology

“…Peptide antisera 128 and 330 against BRF and antiserum 4286 against TFIIIC␤ were described and characterized previously (2,6,28,55). Antiserum against the BN51 subunit of PdIII was generously provided by Michael Ittmann (22).…”

Section: Methodsmentioning

confidence: 99%

CK2 Forms a Stable Complex with TFIIIB and Activates RNA Polymerase III Transcription in Human Cells

Johnston

Allison

Morton

et al. 2002

Self Cite

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.Protein kinase CK2 (formerly known as casein kinase II) is ubiquitous and highly conserved in eukaryotes (reviewed in references 1 and 29). It phosphorylates proteins on serine and threonine in both the nucleus and the cytoplasm. CK2 exists as a tetramer, composed of two isozymic catalytic subunits, ␣ and ␣Ј, and two copies of a regulatory ␤ subunit or one copy each of ␤ and the closely related ␤Ј. The CK2␣ and CK2␣Ј subunits are nearly 90% identical and can compensate for each other, but there is also some functional specialization (57, 69). The ␤ subunits allow optimal kinase activity and can regulate substrate specificity; they form a stable dimer linking the two catalytic subunits, which do not contact each other (35).Although its signaling function has long remained obscure, CK2 has been shown recently to form part of the Wnt pathway in both Drosophila and mammals (54, 65). Many studies have found that increases in the level and/or activity of CK2 are associated with cell growth and proliferation (for example, references 3, 4, 7, 25, 30, 34, and 39). Thus, CK2 expression can be increased by mitogens (7, 39), and CK2 is most abundant in cells with high mitotic activity, such as transformed cells and normal colorectal mucosa (34). Indeed, microinjection of CK2 can induce immediate-early gene expression in the absence of growth factors (11). Conversely, inactivation of CK2 by specific antibodies or antisense oligonucleotides can arrest the proliferation of primary human fibroblasts (41,42). Similarly, cell cycle progression is blocked in Saccharomyces ...

Molecular and Cellular Biology

“…Antiserum 4286 was raised by immunizing rabbits with synthetic peptide RPGFSPTSHRLLPTP (human TFIIIC110 residues 897 to 911) coupled to keyhole limpet hemocyanin. Antisera 128 and 330 were raised against residues 533 to 547 and 664 to 677 of human BRF, respectively, and have been described and characterized previously (1,5,29). SL30 is a monoclonal antiserum against TBP that was generously provided by Nouria Hernandez (33).…”

Section: Methodsmentioning

confidence: 99%

Retinoblastoma Protein Disrupts Interactions Required for RNA Polymerase III Transcription

Sutcliffe

Brown

Allison

et al. 2000

Self Cite

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.The retinoblastoma susceptibility gene encodes the important tumor suppressor retinoblastoma protein (RB) (12,15,37,57). Inactivating mutations in this gene are found in many human cancers, including retinoblastomas, many sarcomas, and bladder and small-cell lung carcinomas (12,15,37,57). In a large proportion of other human malignancies the Rb gene is of the wild type, but its function is disrupted. For example, the cyclin-dependent kinases that switch off RB are hyperactive in many tumors (12,15,37,57). Indeed, it has been suggested that the regulatory pathway involving RB may be compromised in all human malignancies (57). It is therefore of considerable importance to obtain a clear understanding of the mechanisms used by RB to influence cellular activity.RB is a highly abundant protein that has been shown to bind and regulate a variety of transcription factors (12,15,37,48). The best-characterized example is the factor E2F, which controls several genes that are transcribed by RNA polymerase (Pol) II (11,14). Indeed, RB was thought for some time to control only Pol II-transcribed genes. However, recent advances have demonstrated that RB can also regulate transcription by Pols I and III (7,58,62). Pol I synthesizes large rRNA, whereas Pol III synthesizes a variety of small stable RNAs, including 5S rRNA and tRNA; together Pols I and III can be responsible for up to 80% of all nuclear transcription (39).Experiments using knockout mice revealed a major role for endogenous RB in regulating Pol III. Primary fibroblasts from Rb Ϫ/Ϫ mice were found to have a fivefold higher Pol III transcriptional activity than equivalent cells from wild-type mice, when assayed in vitro or in vivo (28, 62). Furthermore, overexpression of RB can inhibit Pol III transcription in transfected cells or in a system reconstituted with partially purified factors (8,28,62). This repression involves binding of RB to the Pol III-specific factor TFIIIB (8, 28). TFIIIB is a multisubunit complex which contains the TATA-binding protein (TBP), a TBP-associated factor (TAF) called BRF, and at least one other esse...