Jeffrey D. Parvin

RNA Helicase A Mediates Association of CBP with RNA Polymerase II

The coactivator CBP has been proposed to stimulate the expression of certain signal-dependent genes via its association with RNA polymerase II complexes. Here we show that complex formation between CBP and RNA polymerase II requires RNA helicase A (RHA), a nuclear DNA/RNA helicase that is related to the Drosophila male dosage compensation factor mle. In transient transfection assays, RHA was found to cooperate with CBP in mediating target gene activation via the CAMP responsive factor CREB. As a mutation in RHA that compromised its helicase activity correspondingly reduced CREB-dependent transcription, we propose that RHA may induce local changes in chromatin structure that promote engagement of the transcriptional apparatus on signal responsive promoters.

BRCA1 protein is linked to the RNA polymerase II holoenzyme complex via RNA helicase A

The breast cancer specific tumour suppressor protein, BRCA1 (refs 1,2), activates transcription when linked with a DNA-binding domain and is a component of the RNA polymerase II (Pol II) holoenzyme. We show here that RNA helicase A (RHA) protein links BRCA1 to the holoenzyme complex. The region of BRCA1 which interacts with RHA and, thus, the holoenzyme complex, corresponds to subregions of the BRCT domain of BRCA1 ( ref. 9). This interaction was shown to occur in yeast nuclei, and expression in human cells of a truncated RHA molecule which retains binding to BRCA1 inhibited transcriptional activation mediated by the BRCA1 carboxy terminus. These data are the first to identify a specific protein interaction with the BRCA1 C-terminal domain and are consistent with the model that BRCA1 functions as a transcriptional coactivator.

DNA topology and a minimal set of basal factors for transcription by RNA polymerase II

Immunoglobulin heavy chain (IgH) gene transcription in vitro can be reconstituted with a minimal reaction containing only TATA-binding protein (TBP), TFIIB, and RNA polymerase II (pol II) when the template is negatively supercoiled. Transcription from linear DNA templates containing either the IgH or the adenovirus major late promoters (MLPs) requires in addition TFIIF, TFIIE, TFIIH, and a fraction containing TFIIA and TFIIJ. Promoters vary in their activities in the minimal reaction. Initiation at the adenovirus MLP site was not observed in this reaction, even with templates containing negative superhelical density. When only TBP, TFIIB, and pol II were present in the reaction, the more negatively supercoiled the IgH template DNA was, the more active the transcription. It is suggested that the free energy of supercoiling promotes the formation of an open complex for initiation of transcription by the minimal set of transcription factors.

BRCA1-Dependent Ubiquitination of γ-Tubulin Regulates Centrosome Number

ABSTRACT Proper centrosome duplication and spindle formation are crucial for prevention of chromosomal instability, and BRCA1 plays a role in this process. In this study, transient inhibition of BRCA1 function in cell lines derived from mammary tissue caused rapid amplification and fragmentation of centrosomes. Cell lines tested that were derived from nonmammary tissues did not amplify the centrosome number in this transient assay. We tested whether BRCA1 and its binding partner, BARD1, ubiquitinate centrosome proteins. Results showed that centrosome components, including γ-tubulin, are ubiquitinated by BRCA1/BARD1 in vitro. The in vitro ubiquitination of γ-tubulin was specific, and function of the carboxy terminus was necessary for this reaction; truncated BRCA1 did not ubiquitinate γ-tubulin. BRCA1/BARD1 ubiquitinated lysines 48 and 344 of γ-tubulin in vitro, and expression in cells of γ-tubulin K48R caused a marked amplification of centrosomes. This result supports the notion that the modification of these lysines in living cells is critical in the maintenance of centrosome number. One of the key problems in understanding the biology of BRCA1 has been the identification of a specific target of BRCA1/BARD1 ubiquitination and its effect on mammary cell biology. The results of this study identify a ubiquitination target and suggest a biological impact important in the etiology of breast cancer.

The multiple nuclear functions of BRCA1: transcription, ubiquitination and DNA repair.

Interest in BRCA1 stems from its role as a tumour suppressor in breast and ovarian cancer. Intensive research in BRCA1 has revealed little about its specific role in cancer; rather, this protein has been implicated in a multitude of important cellular processes. The diverse biochemical activities of BRCA1 combine to protect the genome from damage. New data reveal that BRCA1 transcriptionally regulates some DNA-repair genes, and, in addition, new roles for BRCA1 have been identified in heterochromatin formation on the X chromosome, double-strand-break repair, and ubiquitination. These diverse activities of BRCA1 may be linked in a single pathway, or BRCA1 might function in multiple nuclear processes.

An eukaryotic RuvB-like protein (RUVBL1) essential for growth

A human protein (RUVBL1), consisting of 456 amino acids (50 kDa) and highly homologous to RuvB, was identified by using the 14-kDa subunit of replication protein A (hsRPA3) as bait in a yeast two-hybrid system. RuvB is a bacterial protein involved in genetic recombination that bears structural similarity to subunits of the RF-C clamp loader family of proteins. Fluorescence in situhybridization analysis demonstrated that the RUVBL1 gene is located at 3q21, a region with frequent rearrangements in different types of leukemia and solid tumors. RUVBL1 co-immunoprecipitated with at least three other unidentified cellular proteins and was detected in the RNA polymerase II holoenzyme complex purified over multiple chromatographic steps. In addition, two yeast homologs, scRUVBL1 and scRUVBL2 with 70 and 42% identity to RUVBL1, respectively, were revealed by screening the complete Saccharomyces cerevisiae genome sequence. Yeast with a null mutation in scRUVBL1 was nonviable. Thus RUVBL1 is an eukaryotic member of the RuvB/clamp loader family of structurally related proteins from bacteria and eukaryotes that is essential for viability of yeast.

Degradation of Cdt1 during S phase is Skp2-independent and is required for efficient progression of mammalian cells through S phase.

Previous reports have shown that the N terminus of Cdt1 is required for its degradation during S phase (Li, X., Zhao, Q., Liao, R., Sun, P., and Wu, X. (2003) J. Biol. Chem. 278, 30854–30858; Nishitani, H., Lygerou, Z., and Nishimoto, T. (2004) J. Biol. Chem. 279, 30807–30816). The stabilization was attributed to deletion of the cyclin binding motif (Cy motif), which is required for its phosphorylation by cyclin-dependent kinases. Phosphorylated Cdt1 is subsequently recognized by the F-box protein Skp2 and targeted for proteasomal mediated degradation. Using phosphopeptide mapping and mutagenesis studies, we found that threonine 29 within the N terminus of Cdt1 is phosphorylated by Cdk2 and required for interaction with Skp2. However, threonine 29 and the Cy motif are not necessary for proteolysis of Cdt1 during S phase. Mutants of Cdt1 that do not stably associate with Skp2 or cyclins are still degraded in S phase to the same extent as wild type Cdt1, indicating that other determinants within the N terminus of Cdt1 are required for degrading Cdt1. We localized the region necessary for Cdt1 degradation to the first 32 residues. Overexpression of stable forms of Cdt1 significantly delayed entry into and completion of S phase, suggesting that failure to degrade Cdt1 prevents normal progression through S phase. In contrast, Cdt1 mutants that fail to interact with Skp2 and cyclins progress through S phase with similar kinetics as wild type Cdt1 but stimulate the re-replication caused by overexpressing Cdt1. Therefore, a Skp2-independent pathway that requires the N-terminal 32 residues of Cdt1 is critical for the degradation of Cdt1 in S phase, and this degradation is necessary for the optimum progression of cells through S phase.

Promoter specificity of basal transcription factors

Regulation of expression of protein-encoding genes in eukaryotes is frequently mediated by sequence-specific transcription factors that control the activities of the basal factors and RNA polymerase II. Basal factors have been considered to be essential for all polymerase II promoters. Studies of the basal factor requirements for transcription from the immunoglobulin heavy chain gene (IgH) core promoter and the adenovirus major late gene core promoter (MLP) suggest that this paradigm is too simple. Basal transcription from the IgH promoter was reconstituted by TFIID, TFIIB, TFIIF, and polymerase, whereas basal transcription from the MLP is highly dependent upon TFIIE in addition to the above factors. Two novel protein activities, referred to as 700 kd and 90 kd, further stimulated the basal reaction from the MLP. Thus, these data indicate that not all basal factors are in fact general.

Centrosomal Microtubule Nucleation Activity Is Inhibited by BRCA1-Dependent Ubiquitination

ABSTRACT In this study we find that the function of BRCA1 inhibits the microtubule nucleation function of centrosomes. In particular, cells in early S phase have quiescent centrosomes due to BRCA1 activity, which inhibits the association of γ-tubulin with centrosomes. We find that modification of either of two specific lysine residues (Lys-48 and Lys-344) of γ-tubulin, a known substrate for BRCA1-dependent ubiquitination activity, led to centrosome hyperactivity. Interestingly, mutation of γ-tubulin lysine 344 had a minimal effect on centrosome number but a profound effect on microtubule nucleation function, indicating that the processes regulating centrosome duplication and microtubule nucleation are distinct. Using an in vitro aster formation assay, we found that BRCA1-dependent ubiquitination activity directly inhibits microtubule nucleation by centrosomes. Mutant BRCA1 protein that was inactive as a ubiquitin ligase did not inhibit aster formation by the centrosome. Further, a BRCA1 carboxy-terminal truncation mutant that was an active ubiquitin ligase lacked domains critical for the inhibition of centrosome function. These experiments reveal an important new functional assay regulated by the BRCA1-dependent ubiquitin ligase, and the results suggest that the loss of this BRCA1 activity could cause the centrosome hypertrophy and subsequent aneuploidy typically found in breast cancers.

DNA topoisomerase II|[alpha]| is required for RNA polymerase II transcription on chromatin templates

In the nucleus of the cell, core RNA polymerase II (pol II) is associated with a large complex called the pol II holoenzyme (holo-pol). Transcription by core pol II in vitro on nucleosomal templates is repressed compared with that on templates of histone-free naked DNA. We found that the transcriptional activity of holo-pol, in contrast to that of core pol II, is not markedly repressed on chromatin templates. We refer to this property of holo-pol as chromatin-dependent coactivation (CDC). Here we show that DNA topoisomerase IIα is associated with the holo-pol and is a required component of CDC. Etoposide and ICRF-193, specific inhibitors of topoisomerase II, blocked transcription on chromatin templates, but did not affect transcription on naked templates. Addition of purified topoisomerase IIα reconstituted CDC activity in reactions with core pol II. These findings suggest that transcription on chromatin templates results in the accumulation of superhelical tension, making the relaxation activity of topoisomerase II essential for productive RNA synthesis on nucleosomal DNA.