Clustering phenotype populations by genome-wide RNAi and multiparametric imaging.

Genetic screens for phenotypic similarity have made key contributions to associating genes with biological processes. With RNA interference (RNAi), highly parallel phenotyping of loss-of-function effects in cells has become feasible. One of the current challenges however is the computational categorization of visual phenotypes and the prediction of biological function and processes. In this study, we describe a combined computational and experimental approach to discover novel gene functions and explore functional relationships. We performed a genome-wide RNAi screen in human cells and used quantitative descriptors derived from high-throughput imaging to generate multiparametric phenotypic profiles. We show that profiles predicted functions of genes by phenotypic similarity. Specifically, we examined several candidates including the largely uncharacterized gene DONSON, which shared phenotype similarity with known factors of DNA damage response (DDR) and genomic integrity. Experimental evidence supports that DONSON is a novel centrosomal protein required for DDR signalling and genomic integrity. Multiparametric phenotyping by automated imaging and computational annotation is a powerful method for functional discovery and mapping the landscape of phenotypic responses to cellular perturbations.

Acetylation of cyclin T1 regulates the equilibrium between active and inactive P-TEFb in cells.

The elongation competence of the RNA polymerase II complex is critically dependent on the positive transcription elongation factor b (P-TEFb). P-TEFb exists in two forms in cells, an active form composed of cyclin T1 and CDK9 and an inactive form, in which cyclin T1/CDK9 is sequestered by Hexim1 and 7SK snRNA. Here, we report that partitioning of active and inactive P-TEFb is regulated by acetylation of cyclin T1. Cyclin T1 acetylation triggers dissociation of Hexim1 and 7SK snRNA from cyclin T1/CDK9 and activates the transcriptional activity of P-TEFb. This activation is lost in P-TEFb complexes containing cyclin T1 that can no longer be acetylated. An acetylation-deficient cyclin T1 mutant dominantly suppresses NF-kappaB-mediated activation of the interleukin-8 promoter but continues to synergize normally with the HIV Tat protein to transactivate the HIV long terminal repeat. These findings support the model that acetylation of cyclin T1 serves as a physiological switch that liberates P-TEFb from its endogenous inhibitors Hexim1 and 7SK snRNA, but is not required for the cooperative action with HIV Tat.

Recruitment and activation of RSK2 by HIV-1 Tat.

The transcriptional activity of the integrated HIV provirus is dependent on the chromatin organization of the viral promoter and the transactivator Tat. Tat recruits the cellular pTEFb complex and interacts with several chromatin-modifying enzymes, including the histone acetyltransferases p300 and PCAF. Here, we examined the interaction of Tat with activation-dependent histone kinases, including the p90 ribosomal S6 kinase 2 (RSK2). Dominant-negative RSK2 and treatment with a small-molecule inhibitor of RSK2 kinase activity inhibited the transcriptional activity of Tat, indicating that RSK2 is important for Tat function. Reconstitution of RSK2 in cells from subjects with a genetic defect in RSK2 expression (Coffin-Lowry syndrome) enhanced Tat transactivation. Tat interacted with RSK2 and activated RSK2 kinase activity in cells. Both properties were lost in a mutant Tat protein (F38A) that is deficient in HIV transactivation. Our data identify a novel reciprocal regulation of Tat and RSK2 function, which might serve to induce early changes in the chromatin organization of the HIV LTR.

SIRT1 regulates HIV transcription via Tat deacetylation.

The human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional coactivator p300, a necessary step in Tat-mediated transactivation. We report here that Tat is deacetylated by human sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent class III protein deacetylase in vitro and in vivo. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV promoter. Conversely, knockdown of SIRT1 via small interfering RNAs or treatment with a novel small molecule inhibitor of the SIRT1 deacetylase activity inhibit Tat-mediated transactivation of the HIV long terminal repeat. Tat transactivation is defective in SIRT1-null mouse embryonic fibroblasts and can be rescued by expression of SIRT1. These results support a model in which cycles of Tat acetylation and deacetylation regulate HIV transcription. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional coactivator during Tat transactivation.

Release and intercellular transfer of cell surface CD81 via microparticles.

The human tetraspan molecule CD81 is a coreceptor in B and T cell activation and a candidate receptor for hepatitis C virus infection. We examined the surface expression of CD81 on B and T lymphocytes by quantitative flow cytometry. Upon cellular activation, CD81 surface levels were rapidly reduced. This reduction occurred as early as 1 h after activation and was linked to the release of CD81-positive microparticles into the cell culture medium. CD81 mRNA levels were not affected early after activation, but the release of CD81-positive microparticles was rapidly enhanced. In addition, intercellular transfer of CD81 was observed upon coculture of CD81-positive donor cells (Jurkat T cell line) with CD81-negative acceptor cells (U937 promonocytic cell line). This transfer was rapidly increased upon T cell activation, coinciding with enhanced CD81 release from activated Jurkat cells. We propose that the release and intercellular trafficking of CD81-positive microparticles regulate the expression of CD81 surface receptors in lymphocytes and play a role in the immune response during infections.

Transcriptional synergy between Tat and PCAF is dependent on the binding of acetylated Tat to the PCAF bromodomain.

The human immunodeficiency virus (HIV) Tat protein plays an essential role in promoting efficient transcriptional elongation of viral transcripts. We report that the transcriptional co-activator PCAF and Tat interact and synergize to activate the HIV promoter. The binding of Tat and PCAF in vitro and in vivo is dependent on the acetylated state of Lys50 of Tat and on the PCAF bromodomain. Structural analysis of the acetylated Tat peptide bound to the PCAF bromodomain defined amino acids Y47 and R53 in Tat and V763, Y802, and Y809 in PCAF as critical interaction points between the two proteins. Mutation of each of these residues in either Tat or PCAF inhibited in a cumulative manner the Tat-PCAF interaction in vitro and in vivo, and abrogated the synergistic activation of the HIV promoter by both proteins. These observations demonstrate that acetylation of Tat establishes a novel protein-protein interaction domain at the surface of Tat that is necessary for the transcriptional activation of the HIV promoter.