Brendan Bell

The N-terminal domain of human TAFII68 displays transactivation and oncogenic properties.

In Ewing tumor, the (11;22) chromosomal translocation produces a chimeric molecule composed of the amino-terminal domain of EWS fused to the carboxyl-terminal DNA-binding domain of FLI-1. Previously, we have identified a novel protein TAFII68, which is highly similar to EWS and another closely related protein TLS (also called FUS). We demonstrate that the N-terminus of TAFII68 efficiently stimulates transcription when fused to two different DNA binding domains and that overexpression of TAFII68-FLI-1 chimeras in NIH3T3 cells leads to oncogenic transformation. We have also investigated the molecular mechanisms which could account for the transcriptional activation and the oncogenic transformation potential of the N-termini of TAFII68 and EWS. Thus, we have tested whether the artificial recruitment of components of the preinitiation complex (PIC) or a histone acetyltransferase (HAT) could bypass the requirement for the activation domains of either EWS or TAFII68. Recruitment of individual components of the transcription machinery or the GCN5 HAT is not sufficient to promote activation from FLI-1 responsive genes either in transfection experiments or in oncogenic transformation assays. These results suggest that the TAFII68 or EWS activation domains enhance a step after PIC formation in the transcriptional activation process.

Cancer-Associated Perturbations in Alternative Pre-messenger RNA Splicing

For most of our 25,000 genes, the removal of introns by pre-messenger RNA (pre-mRNA) splicing represents an essential step toward the production of functional messenger RNAs (mRNAs). Alternative splicing of a single pre-mRNA results in the production of different mRNAs. Although complex organisms use alternative splicing to expand protein function and phenotypic diversity, patterns of alternative splicing are often altered in cancer cells. Alternative splicing contributes to tumorigenesis by producing splice isoforms that can stimulate cell proliferation and cell migration or induce resistance to apoptosis and anticancer agents. Cancer-specific changes in splicing profiles can occur through mutations that are affecting splice sites and splicing control elements, and also by alterations in the expression of proteins that control splicing decisions. Recent progress in global approaches that interrogate splicing diversity should help to obtain specific splicing signatures for cancer types. The development of innovative approaches for annotating and reprogramming splicing events will more fully establish the essential contribution of alternative splicing to the biology of cancer and will hopefully provide novel targets and anticancer strategies. Metazoan genes are usually made up of several exons interrupted by introns. The introns are removed from the pre-mRNA by RNA splicing. In conjunction with other maturation steps, such as capping and polyadenylation, the spliced mRNA is then transported to the cytoplasm to be translated into a functional protein. The basic mechanism of splicing requires accurate recognition of each extremity of each intron by the spliceosome. Introns are identified by the binding of U1 snRNP to the 5 splice site and the U2AF65/U2AF35 complex to the 3 splice site. Following these interactions, other proteins and snRNPs are recruited to generate the complete spliceosomal complex needed to excise the intron. While many introns are constitutively removed by the spliceosome, other splice junctions are not used systematically, generating the phenomenon of alternative splicing. Alternative splicing is therefore the process by which a single species of pre-mRNA can be matured to produce different mRNA molecules (Fig.xa01). Depending on the number and types of alternative splicing events, a pre-mRNA can generate from two to several thousands different mRNAs leading to the production of a corresponding number of proteins. It is now believed that the expression of at least 70xa0% of human genes is subjected to alternative splicing, implying an enormous contribution to proteomic diversity, and by extension, to the development and the evolution of complex animals. Defects in splicing have been associated with human diseases (Caceres and Kornblihtt, Trends Genet 18(4):186-93, 2002, Cartegni et al., Nat Rev Genet 3(4):285-98, 2002, Pagani and Baralle, Nat Rev Genet 5(5):389-96, 2004), including cancer (Brinkman, Clin Biochem 37(7):584-94, 2004, Venables, Bioessays 28(4):378-86, 2006, Srebrow and Kornblihtt, J Cell Sci 119(Pt 13):2635-2641, 2006, Revil et al., Bull Cancer 93(9):909-919, 2006, Venables, Transworld Res Network, 2006, Pajares et al., Lancet Oncol 8(4):349-57, 2007, Skotheim and Nees, Int J Biochem Cell Biol 39:1432-1449, 2007). Numerous studies have now confirmed the existence of specific differences in the alternative splicing profiles between normal and cancer tissues. Although there are a few cases where specific mutations are the primary cause for these changes, global alterations in alternative splicing in cancer cells may be primarily derived from changes in the expression of RNA-binding proteins that control splice site selection. Overall, these cancer-specific differences in alternative splicing offer an immense potential to improve the diagnosis and the prognosis of cancer. This review will focus on the functional impact of cancer-associated alternative splicing variants, the molecular determinants that alter the splicing decisions in cancer cells, and future therapeutic strategies.

CTGC motifs within the HIV core promoter specify Tat-responsive pre-initiation complexes

BackgroundHIV latency is an obstacle for the eradication of HIV from infected individuals. Stable post-integration latency is controlled principally at the level of transcription. The HIV trans-activating protein, Tat, plays a key function in enhancing HIV transcriptional elongation. The HIV core promoter is specifically required for Tat-mediated trans-activation of HIV transcription. In addition, the HIV core promoter has been shown to be a potential anti-HIV drug target. Despite the pivotal role of the HIV core promoter in the control of HIV gene expression, the molecular mechanisms that couple Tat function specifically to the HIV core promoter remain unknown.ResultsUsing electrophoretic mobility shift assays (EMSAs), the TATA box and adjacent sequences of HIV essential for Tat trans-activation were shown to form specific complexes with nuclear extracts from peripheral blood mononuclear cells, as well as from HeLa cells. These complexes, termed pre-initiation complexes of HIV (PICH), were distinct in composition and DNA binding specificity from those of prototypical eukaryotic TATA box regions such as Adenovirus major late promoter (AdMLP) or the hsp70 promoter. PICH contained basal transcription factors including TATA-binding protein and TFIIA. A mutational analysis revealed that CTGC motifs flanking the HIV TATA box are required for Tat trans-activation in living cells and correct PICH formation in vitro. The binding of known core promoter binding proteins AP-4 and USF-1 was found to be dispensable for Tat function. TAR RNA prevented stable binding of PICH-2, a complex that contains the general transcription factor TFIIA, to the HIV core promoter. The impact of TAR on PICH-2 specifically required its bulge sequence that is also known to interact with Tat.ConclusionOur data reveal that CTGC DNA motifs flanking the HIV TATA box are required for correct formation of specific pre-initiation complexes in vitro and that these motifs are also required for Tat trans-activation in living cells. The impact of TAR RNA on PICH-2 stability provides a mechanistic link by which pre-initiation complex dynamics could be coupled to the formation of the nascent transcript by the elongating transcription complex. Together, these findings shed new light on the mechanisms by which the HIV core promoter specifically responds to Tat to activate HIV gene expression.

HMGA1 directly interacts with TAR to modulate basal and Tat-dependent HIV transcription

The transactivating response element (TAR) of human immunodeficiency virus 1 (HIV-1) is essential for promoter transactivation by the viral transactivator of transcription (Tat). The Tat-TAR interaction thereby recruits active positive transcription elongation factor b (P-TEFb) from its inactive, 7SK/HEXIM1-bound form, leading to efficient viral transcription. Here, we show that the 7SK RNA-associating chromatin regulator HMGA1 can specifically bind to the HIV-1 TAR element and that 7SK RNA can thereby compete with TAR. The HMGA1-binding interface of TAR is located within the binding site for Tat and other cellular activators, and we further provide evidence for competition between HMGA1 and Tat for TAR-binding. HMGA1 negatively influences the expression of a HIV-1 promoter-driven reporter in a TAR-dependent manner, both in the presence and in the absence of Tat. The overexpression of the HMGA1-binding substructure of 7SK RNA results in a TAR-dependent gain of HIV-1 promoter activity similar to the effect of the shRNA-mediated knockdown of HMGA1. Our results support a model in which the HMGA1/TAR interaction prevents the binding of transcription-activating cellular co-factors and Tat, subsequently leading to reduced HIV-1 transcription.

TAF6δ Controls Apoptosis and Gene Expression in the Absence of p53

Background Life and death decisions of metazoan cells hinge on the balance between the expression of pro- versus anti-apoptotic gene products. The general RNA polymerase II transcription factor, TFIID, plays a central role in the regulation of gene expression through its core promoter recognition and co-activator functions. The core TFIID subunit TAF6 acts in vitro as an essential co-activator of transcription for the p53 tumor suppressor protein. We previously identified a splice variant of TAF6, termed TAF6δ that can be induced during apoptosis. Methodology/Principal Findings To elucidate the impact of TAF6δ on cell death and gene expression, we have employed modified antisense oligonucleotides to enforce expression of endogenous TAF6δ. The induction of endogenous TAF6δ triggered apoptosis in tumor cell lines, including cells devoid of p53. Microarray experiments revealed that TAF6δ activates gene expression independently of cellular p53 status. Conclusions Our data define TAF6δ as a pivotal node in a signaling pathway that controls gene expression programs and apoptosis in the absence of p53.

Determining the impact of alternative splicing events on transcriptome dynamics

BackgroundThe complete sequencing of the human genome and its subsequent analysis revealed a predominant role for alternative splicing in the generation of proteome diversity. Splice switching oligonucleotides (SSOs) are a powerful and specific tool to experimentally control alternative splicing of endogenous messenger RNAs in living cells. SSOs also have therapeutic potential to treat diseases that are caused by aberrant splicing. The assignment of biological roles to alternative splicing events of currently unknown function promises to provide a largely untapped source of potential new therapeutic targets. Here we have developed a protocol that combines high sensitivity microarrays with the transfection of SSOs to monitor global changes in gene expression downstream of alternate, endogenous splice events.ResultsWhen applied to a well-characterized splicing event in the Bcl-x gene, the application of high sensitivity microarrays revealed a link between the induction of the Bcl-xS isoform and the repression of genes involved in protein synthesis.ConclusionThe strategy introduced herein provides a useful approach to define the biological impact of any given alternative splicing event on global gene expression patterns. Furthermore, our data provide the first link between Bcl-xS expression and the repression of ribosomal protein gene expression.

TAF6δ orchestrates an apoptotic transcriptome profile and interacts functionally with p53

BackgroundTFIID is a multiprotein complex that plays a pivotal role in the regulation of RNA polymerase II (Pol II) transcription owing to its core promoter recognition and co-activator functions. TAF6 is a core TFIID subunit whose splice variants include the major TAF6α isoform that is ubiquitously expressed, and the inducible TAF6δ. In contrast to TAF6α, TAF6δ is a pro-apoptotic isoform with a 10 amino acid deletion in its histone fold domain that abolishes its interaction with TAF9. TAF6δ expression can dictate life versus death decisions of human cells.ResultsHere we define the impact of endogenous TAF6δ expression on the global transcriptome landscape. TAF6δ was found to orchestrate a transcription profile that included statistically significant enrichment of genes of apoptotic function. Interestingly, gene expression patterns controlled by TAF6δ share similarities with, but are not equivalent to, those reported to change following TAF9 and/or TAF9b depletion. Finally, because TAF6δ regulates certain p53 target genes, we tested and demonstrated a physical and functional interaction between TAF6δ and p53.ConclusionTogether our data define a TAF6δ-driven apoptotic gene expression program and show crosstalk between the p53 and TAF6δ pathways.

Modulation of the splicing regulatory function of SRSF10 by a novel compound that impairs HIV-1 replication

Abstract We recently identified the 4-pyridinone-benzisothiazole carboxamide compound 1C8 as displaying strong anti-HIV-1 potency against a variety of clinical strains in vitro. Here we show that 1C8 decreases the expression of HIV-1 and alters splicing events involved in the production of HIV-1 mRNAs. Although 1C8 was designed to be a structural mimic of the fused tetracyclic indole compound IDC16 that targets SRSF1, it did not affect the splice site shifting activity of SRSF1. Instead, 1C8 altered splicing regulation mediated by SRSF10. Depleting SRSF10 by RNA interference affected viral splicing and, like 1C8, decreased expression of Tat, Gag and Env. Incubating cells with 1C8 promoted the dephosphorylation of SRSF10 and increased its interaction with hTra2β, a protein previously implicated in the control of HIV-1 RNA splicing. While 1C8 affects the alternative splicing of cellular transcripts controlled by SRSF10 and hTra2β, concentrations greater than those needed to inhibit HIV-1 replication were required to elicit significant alterations. Thus, the ability of 1C8 to alter the SRSF10-dependent splicing of HIV-1 transcripts, with minor effects on cellular splicing, supports the view that SRSF10 may be used as a target for the development of new anti-viral agents.

Alternative Splicing of TAF6: Downstream Transcriptome Impacts and Upstream RNA Splice Control Elements

The TAF6δ pathway of apoptosis can dictate life versus death decisions independently of the status of p53 tumor suppressor. TAF6δ is an inducible pro-apoptotic subunit of the general RNA polymerase II (Pol II) transcription factor TFIID. Alternative splice site choice of TAF6δ has been shown to be a pivotal event in triggering death via the TAF6δ pathway, yet nothing is currently known about the mechanisms that promote TAF6δ splicing. Furthermore the transcriptome impact of the gain of function of TAF6δ versus the loss of function of the major TAF6α splice form remains undefined. Here we employ comparative microarray analysis to show that TAF6δ drives a transcriptome profile distinct from that resulting from depletion of TAF6α. To define the cis-acting RNA elements responsible for TAF6δ alternative splicing we performed a mutational analysis of a TAF6 minigene system. The data point to several new RNA elements that can modulate TAF6δ and also reveal a role for RNA secondary structure in the selection of TAF6δ.

Probing Endogenous RNA Polymerase II Pre-initiation Complexes by Electrophoretic Mobility Shift Assay

RNA polymerase II (Pol II) plays a crucial role in eukaryotic biology since it is necessary for the expression of all protein-coding genes as well as most microRNAs and several small nuclear RNAs. Pol II is specifically recruited to core promoter DNA via its association with general transcription factors (GTFs) that possess DNA binding activity such as TFIID, TFIIA, and TFIIB. The large multi-protein assemblies of Pol II together with the GTFs required for productive transcription are termed pre-initiation complexes (PICs). To date, studies of the interaction of PICs with promoter DNA have relied on the use of purified or recombinant GTFs. Recent findings have demonstrated an astonishing diversity in the function of core promoters as well as in the protein composition of PICs. The currently known subset of GTFs alone cannot account for observed PIC and core promoter diversity. In order to identify the full complement of factors that impart PIC specificity, techniques to analyze the DNA binding of endogenous PIC are essential. Analysis of endogenous PIC formation has remained out of reach due to technical hurdles presumably including the large size of endogenous PIC, their highly dynamic association with core promoters, and the complex topology of DNA bound to PIC. We have optimized electrophoretic mobility shift assays (EMSAs) to achieve the detection of endogenous Pol II PIC from nuclear extracts of human cells. Here, we provide a robust and sensitive EMSA method for the analysis of endogenous Pol II PICs.