When noise makes music: HIV reactivation with transcriptional noise enhancers

Reactivating latent HIV is key to depleting the virus reservoir in AIDS patients. A recent paper has described the rationale for and discovery of a new class of drugs - transcriptional noise enhancers - that can synergize with conventional transcription activators to more effectively reactivate latently infected T cells. As well as describing a promising new strategy in the bid to find a cure for AIDS, this study more broadly highlights the utility of exploring drug combinations in treatment of human disease

BRCA1 as tumor suppressor: lord without its RING?

BRCA1 is a tumor suppressor with critical roles in the maintenance of genomic stability. It encodes a large protein with an amino-terminal RING domain that possesses ubiquitin-ligase activity. Given the occurrence of numerous cancer-causing mutations within its RING domain, investigators have long suspected that BRCA1's ubiquitin ligase is important for its tumor suppression and DNA repair activities. Using genetically engineered mouse models, two recent studies shed light on this age-old hypothesis

Multiple Tumor Suppressor Pathways Negatively Regulate Telomerase

AbstractTelomerase expression is repressed in most somatic cells but is observed in stem cells and a high percentage of human cancers and has been hypothesized to contribute to tumorigenesis and maintenance of stem cell states. To explore telomerase regulation, we employed a general genetic screen to identify negative regulators of hTERT. We discovered three tumor suppressor/oncogene pathways involved in hTERT repression. One, the Mad1/c-Myc pathway, had been previously implicated in hTERT regulation. The second, SIP1, a transcriptional target of the TGF-β pathway, mediates the TGF-β regulated repression of hTERT. The third, the tumor suppressor Menin, is a direct repressor of hTERT. Depleting Menin immortalizes primary human fibroblasts and causes a transformation phenotype when coupled with expression of SV40 Large and Small T antigen and oncogenic ras. These studies suggest that multiple tumor suppressor/oncogene pathways coordinately repress hTERT expression and imply that telomerase is reactivated in human tumors through oncogenic mutations

DNA polymerase ϵ links the DNA replication machinery to the S phase checkpoint

AbstractInhibition of DNA synthesis induces transcription of DNA damage -inducible genes and prevents mitotic entry through the action of the S phase checkpoint. We have isolated a mutant, dun2, defective for both of these responses. DUN2 is identical to POL2, encoding DNA polymerase ϵ (pol ϵ). Unlike sadl mutants defective for multiple cell cycle checkpoints, pol2 mutants are defective only for the S phase checkpoint and the activation of DUNI kinase necessary for the transcriptional response to damage. Interallelic complementation and mutation analysis indicate that pol ϵ contains two separable essential domains, an N-terminal polymerase domain and a C-terminal checkpoint domain unique to ϵ polymerases. We propose that DNA pol ϵ acts as a sensor of DNA replication that coordinates the transcriptional and cell cycle responses to replication blocks

Mice Lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control

Abstractp21CIP11WAF1 is a CDK Inhibitor regulated by the tumor suppressor p53 and is hypothesized to mediate G1 arrest. p53 has been suggested to derive anti-oncogenic properties from this relationship. To test these notions, we created mice lacking p21CIP1/WAF1. They develop normally and (unlike p53−/− mice) have not developed spontaneous malignancies during 7 months of observation. Nonetheless, p21−/− embryonic fibroblasts are significantly deficient in their ability to arrest in G1 In response to DNA damage and nucleotide pool perturbation. p21−/− cells also exhibit a significant growth alteration in vitro, achieving a saturation density as high as that observed In p53−/− cells. In contrast, other aspects of p53 function, such as thymocytic apoptosis and the mitotic spindle checkpoint, appear normal. These results establish the role of p21CIP1/WAF1 in the G1 checkpoint, but suggest that the antiapoptotic and the anti-oncogenic effects of p53 are more complex

The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes

AbstractBackground: Modern biological research is highly dependent upon recombinant DNA technology. Conventional cloning methods are time-consuming and lack uniformity. Thus, biological research is in great need of new techniques to rapidly, systematically and uniformly manipulate the large sets of genes currently available from genome projects.Results: We describe a series of new cloning methods that facilitate the rapid and systematic construction of recombinant DNA molecules. The central cloning method is named the univector plasmid-fusion system (UPS). The UPS uses Cre–lox site-specific recombination to catalyze plasmid fusion between the univector – a plasmid containing the gene of interest – and host vectors containing regulatory information. Fusion events are genetically selected and place the gene under the control of new regulatory elements. A second UPS-related method allows for the precise transfer of coding sequences only from the univector into a host vector. The UPS eliminates the need for restriction enzymes, DNA ligases and many in vitro manipulations required for subcloning, and allows for the rapid construction of multiple constructs for expression in multiple organisms. We demonstrate that UPS can also be used to transfer whole libraries into new vectors. Additional adaptations are described, including directional PCR cloning and the generation of 3′ end gene fusions using homologous recombination in Escherichia coli.Conclusions: Together, these recombination-based cloning methods constitute a new comprehensive approach for the rapid and efficient generation of recombinant DNA that can be used for parallel processing of large gene sets, a feature that will facilitate future genomic analysis

The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons

Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small and a facile technology to characterize them is lacking. Using a strategy combining Global Protein Stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Using CRISPR screening, we established that the stability of many proteins is regulated through degrons located at their C-terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complexes adaptors that regulate C-terminal degrons including six CRL2 and two CRL4 complexes and computationally implicate multiple non-CRLs in end recognition. Human proteome analysis revealed that the C-termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3 ligase-dependent modulation of proteome composition. Thus, we propose that a series of ‘C-end rules’ operate to govern protein stability and shape the eukaryotic proteome

Comprehensive identification of host modulators of HIV-1 replication using multiple orthologous RNAi reagents

RNAi screens have implicated hundreds of host proteins as HIV-1 dependency factors (HDFs). While informative, these early studies overlap poorly due to false positives and false negatives. To ameliorate these issues, we combined information from the existing HDF screens together with new screens performed with multiple orthologous RNAi reagents (MORR). In addition to being traditionally validated, the MORR screens and the historical HDF screens were quantitatively integrated by the adaptation of an established analysis program, RIGER, for the collective interpretation of each gene\u27s phenotypic significance. False positives were addressed by the removal of poorly expressed candidates through gene expression filtering, as well as with GESS, which identifies off-target effects. This workflow produced a quantitatively integrated network of genes that modulate HIV-1 replication. We further investigated the roles of GOLGI49, SEC13, and COG in HIV-1 replication. Collectively, the MORR-RIGER method minimized the caveats of RNAi screening and improved our understanding of HIV-1-host cell interactions