Positive Transcription Elongation Factor Complex Research Articles

VIP152 is a selective CDK9 inhibitor with pre-clinical in vitro and in vivo efficacy in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is effectively treated with targeted therapies including Bruton tyrosine kinase inhibitors and BCL2 antagonists. When these become ineffective, treatment options are limited. Positive transcription elongation factor complex (P-TEFb), a heterodimeric protein complex composed of cyclin dependent kinase 9 (CDK9) and cyclin T1, functions to regulate short half-life transcripts by phosphorylation of RNA Polymerase II (POLII). These transcripts are frequently dysregulated in hematologic malignancies; however, therapies targeting inhibition of P-TEFb have not yet achieved approval for cancer treatment. VIP152 kinome profiling revealed CDK9 as the main enzyme inhibited at 100 nM, with over a 10-fold increase in potency compared with other inhibitors currently in development for this target. VIP152 induced cell death in CLL cell lines and primary patient samples. Transcriptome analysis revealed inhibition of RNA degradation through the AU-Rich Element (ARE) dysregulation. Mechanistically, VIP152 inhibits the assembly of P-TEFb onto the transcription machinery and disturbs binding partners. Finally, immune competent mice engrafted with CLL-like cells of Eµ-MTCP1 over-expressing mice and treated with VIP152 demonstrated reduced disease burden and improvement in overall survival compared to vehicle-treated mice. These data suggest that VIP152 is a highly selective inhibitor of CDK9 that represents an attractive new therapy for CLL.

JMJD5 couples with CDK9 to release the paused RNA polymerase II

More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations

Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase-the positive transcription elongation factor complex (P-TEFb)-from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype.

Abstract 5137: Parallel approaches to the discovery of novel BRD4 inhibitors

Abstract Epigenetic signalling is essential for the regulation of gene expression and cellular development, and disruption of these pathways has been identified in a wide range of tumours. The ϵ-N-acetylation of lysine residues (Kac) within histone tails is one of the most abundant epigenetic modifications and bromodomain containing proteins (BRD) represent an important class of reader protein that recognize and bind such residues. BRD4 is a critical mediator of transcription, functioning to recruit the positive transcription elongation factor complex (P-TEFb) and resulting in increased expression of growth-promoting genes. Inhibition of BRD4 has been shown to result in tumour growth inhibition both in vitro and in vivo. In addition, BRD4 has been shown to be a proto-oncogene which is mutated by chromosomal translocations in the rare form of NUT midline carcinoma. Although the interaction between bromodomains and acetylated histone represents a protein-protein interaction, co-crystal structures have demonstrated that Kac is recognized by a central deep hydrophobic cavity, which represents “an attractive druggable pocket”. We performed a virtual screen based on modelling known BRD4 inhibitors in crystal structures of both BRD4 domain 1 and domain 2. The virtual screen used a combination of substructure, shape, electrostatic and docking methods combining the strengths of each of these approaches to select compounds from commercially available sources. In addition, we screened BRD4 binding domain 1 against an Argenta fragment library using surface plasmon resonance. A number of novel hits were identified from these screening approaches (up to 18µM in potency) and crystal structures obtained for key hits. We developed a comprehensive screening cascade validated with tool compounds for profiling hits, starting with an in vitro Brd4 AlphaScreen binding assay and comprising a number of cellular pharmacodynamic assays evaluating effects on established biomarkers of BRD4 inhibition. These screening assays included mRNA analyses for c-myc, p21 and bcl2, a c-myc promoter reporter assay and were confirmed by Western analyses. Phenotypic assays based on growth inhibition of multiple myeloma and acute myeloid leukemia cell lines were also established. Citation Format: David H. Jones, Marcel J. de Groot, Robert Heald, Ebenezer Arhin, Ria Goodwin, Brenda Burton, Jan Kulagowski, David Brown, Steven Irving, Richard Bazin, Deborah Bruce, Rene Devos, Steven Price, Nick Ray, Peter Lockey, John Montana, Mark R. Albertella, Simon R. Green. Parallel approaches to the discovery of novel BRD4 inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5137. doi:10.1158/1538-7445.AM2014-5137

3D structure of HIV-1 tat bound to the host's positive transcription elongation factor complex to assist in development of HIV replication inhibitors

3D structure of HIV-1 tat bound to the host's positive transcription elongation factor complex to assist in development of HIV replication inhibitors

Quantitative Analysis of RNA‐mediated Protein–Protein Interactions in Living Cells by FRET

Specific assembly of ribonucleoprotein complexes is essential in controlling various cellular functions including gene regulation. Diverse scaffolds containing proteins or nucleic acids could play key roles in stabilizing specific ribonucleoprotein complexes by enhancing protein-protein or RNA-protein interactions. One such example is the assembly of active RNA polymerase II transcription elongation complex originating from HIV-1 long terminal repeat promoter that involves HIV-1-encoded Tat protein and viral mRNA structure, trans-activation responsive RNA, and human CyclinT1 which is a subunit of the positive transcription elongation factor complex b. By using genetically encoded fluorescent proteins fused with Tat and human CyclinT1, here we demonstrate that human CyclinT1 was diffused throughout the nucleus and specific interactions between Tat and human CyclinT1 altered the localization of human CyclinT1 to specific nuclear foci. We also found that trans-activation responsive RNA enhanced protein-protein interactions between human CyclinT1 and Tat in living cells. Our results highlights the importance of trans-activation responsive RNA as a scaffold for stable and high affinity assembly of two protein partners to form a regulatory switch essential in HIV-1 gene regulation. RNA-mediated assembly of ribonucleoprotein complexes could be a general mechanism for stable ribonucleoprotein complex formation and a key step in regulating other cellular processes and viral replication. Furthermore, our results suggest that Tat interactions with human CyclinT1 change the nuclear location of positive transcription elongation factor complex b to modulate positive transcription elongation factor complex b function and transcription of cellular genes.

TAR-RNA recognition by a novel cyclic aminoglycoside analogue

The formation of the Tat-protein/TAR-RNA complex is a crucial step in the regulation of human immunodeficiency virus (HIV)-gene expression. To obtain full-length viral transcripts the Tat/TAR complex has to recruit the positive transcription elongation factor complex (P-EFTb), which interacts with TAR through its cyclin T1 (CycT1) component. Mutational studies identified the TAR hexanucleotide loop as a crucial region for contacting CycT1. Interfering with the interaction between the Tat/CycT1 complex and the TAR-RNA is an attractive strategy for the design of anti-HIV drugs. Positively charged molecules, like aminoglycosides or peptidomimetics, bind the TAR-RNA, disrupting the Tat/TAR complex. Here, we investigate the complex between the HIV-2 TAR-RNA and a neooligoaminodeoxysaccharide by NMR spectroscopy. In contrast to other aminoglycosides, this novel aminoglycoside analogue contacts simultaneously the bulge residues required for Tat binding and the A35 residue of the hexanucleotide loop. Upon complex formation, the loop region undergoes profound conformational changes. The novel binding mode, together with the easy accessibility of derivatives for the neooligoaminodeoxysaccharide, could open the way to the design of a new class of TAR-RNA binders, which simultaneously inhibit the formation of both the Tat/TAR binary complex and the Tat/TAR/CycT1 ternary complex by obstructing both the bulge and loop regions of the RNA.

TAR RNA loop: a scaffold for the assembly of a regulatory switch in HIV replication.

Replication of HIV requires the Tat protein, which activates elongation of RNA polymerase II transcription at the HIV-1 promoter by interacting with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex b (P-TEFb). The transactivation domain of Tat binds directly to the CycT1 subunit of P-TEFb and induces loop sequence-specific binding of P-TEFb onto nascent HIV-1 trans-activation responsive region (TAR) RNA. We used systematic RNA-protein photocross-linking, Western blot analysis, and protein footprinting to show that residues 252-260 of CycT1 interact with one side of the TAR RNA loop and enhance interaction of Tat residue K50 to the other side of the loop. Our results show that TAR RNA provides a scaffold for two protein partners to bind and assemble a regulatory switch in HIV replication. RNA-mediated assembly of RNA-protein complexes could be a general mechanism for stable ribonucleoprotein complex formation and a key step in regulating other cellular processes and viral replication.

Specific HIV-1 TAR RNA loop sequence and functional groups are required for human cyclin T1-Tat-TAR ternary complex formation.

Replication of human immunodeficiency virus requires Tat protein which activates elongation of RNA polymerase II transcription at the HIV-1 promoter through interaction with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex (P-TEFb). Tat binds directly through its transactivation domain to the CycT1 subunit of the P-TEFb and induces loop sequence specific binding of the P-TEFb onto nascent HIV-1 TAR RNA. By using a gel electrophoresis method and a comprehensive set of TAR loop mutants, we have identified the sequence and structural determinants for high-affinity CycT1-Tat-TAR ternary complex formation. Our results show that CycT1 and Tat binding to TAR RNA is highly cooperative, and a capacity of 85%, a Hill coefficient of 2.7, and a dissociation constant (K(D)) of 2.45 nM were observed. These results indicate that there are three binding sites on TAR RNA. CycT1 does not bind TAR RNA in the absence of Tat, and Tat binding to TAR, while detectable, is very inefficient in the absence of CycT1. It is conceivable that the CycT1-Tat heterodimer directly binds to TAR RNA in the U-rich RNA bulge region and this binding facilitates the interactions of the CycT1-Tat heterodimer at the other two sites in the RNA loop region. On the basis of our results, we suggest a model where CycT1 interacts with Tat protein and positions the protein complex to make contacts with the G34 region of the loop sequence; G34 is critical for CycT1-Tat binding and forms a C30.G34 base pair. Two functional groups, O6 and N7, at nucleotide positions 32 and 34 in the TAR loop are essential for CycT1-Tat interactions with TAR RNA. The identity of two nucleotides, U31 and G33, is not critical, but they contribute to the stabilization of the RNA-protein complex. The presence of a single-nucleotide bulge of A35 or C35 is essential for distortion of the backbone RNA structure as well as the accessibility of functional groups in the major groove of the double-helical region. CycT1-Tat interaction with TAR RNA represents another example of the flexibility and complexity of RNA structure involved in protein recognition.

HIV-1 TAR RNA Enhances the Interaction between Tat and Cyclin T1

Human immunodeficiency virus, type 1 (HIV-1), Tat activates elongation of RNA polymerase II transcription at the HIV-1 promoter through interaction with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex, P-TEFb. Binding of Tat to CycT1 induces cooperative binding of the P-TEFb complex onto nascent HIV-1 TAR RNA. Here the specific interaction between Tat protein, human cyclin T1, and HIV-1 TAR RNA was analyzed by fluorescence resonance energy transfer, using fluorescein-labeled TAR RNA and a rhodamine-labeled Tat protein synthesized through solid-phase chemistry. We find that CycT1 remodels the structure of Tat to enhance its affinity for TAR RNA and that TAR RNA further enhances the interaction between Tat and CycT1. We conclude that TAR RNA nucleates the formation of the Tat.P-TEFb complex through an induced fit mechanism.