Transcriptional repression of the silent mating-type loci in Saccharomyces cerevisiae requires a cell cycle-dependent establishment step that is commonly assumed to involve DNA replication. Using site-specific recombination, we created a nonreplicating DNA ring in vivo to test directly the role of replication in establishment of silencing. Sir1 was tethered to the ring following excision from the chromosome to activate a dormant silencer. We show here that silencing can be established in DNA that does not replicate. The silenced ring adopted structural features characteristic of bona fide silent chromatin, including an altered level of DNA supercoiling and reduced histone acetylation. In addition, the process required silencing factors Sir2, Sir3, and Sir4 and progression between early S and M phases of the cell cycle. The results indicate that passage of a replication fork is not the cell-cycle event required for establishment of silencing in yeast.

SUMMARY Protein-protein interactions (PPIs) play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable weak PPI detection in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL’s ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a new class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL’s ability to expedite PPI analysis, assess target specificity and cell permeability, and to reveal off-target effects of PPI modifiers should facilitate development of effective, cell permeable PPI therapeutics and elaboration of diverse biological mechanisms.

P53 regulates numerous downstream targets to induce cell cycle arrest, senescence, apoptosis, and DNA repair in response to diverse stresses. Hdm2 and Hdmx are critical negative regulators of P53 because Hdm2 regulates P53 abundance, and both can antagonize P53 transactivation. Modest changes in Hdm2 or Hdmx abundance affect P53 regulation, yet quantitative information regarding their endogenous intracellular concentrations and subcellular distributions during a stress response are lacking. We analyzed these parameters in normal and cancer cells after DNA damage. Our data show that the nuclear abundance of Hdm2 and Hdmx relative to P53 limits P53 activity in cells growing in culture. Upon DNA damage, P53 nuclear abundance increases, whereas Hdm2 and Hdmx stability decreases, which greatly limits their ability to antagonize P53, regardless of their levels. These data indicate that the damage-activated switch in Hdm2 ubiquitin ligase preference from P53 to itself and Hdmx is central to P53 activation. The mechanisms by which P53 is activated after DNA damage have been widely studied (1). Two models that incorporate key roles for damage-activated kinases have emerged to explain how Hdm2-and Hdmx-mediated inhibition of P53 is overcome. The first proposes that activation of the ATM kinase leads to a kinase cascade resulting in phosphorylation of highly conserved serine and threonine residues in P53 within and flanking the Hdm2/ Hdmx-binding region (6). This induces a conformational change in P53, leading to Hdm2 dissociation, P53 stabilization and accumulation, and binding of P53 transcriptional coactivators (7,8). Histone acetyl transferase binding acetylates P53 C-terminal lysines and chromatin and promotes transactivation (9). The situation is likely more complex, because P53 in which highly conserved C-terminal lysines are replaced by arginines has basal and stress-induced stability and activity comparable with wildtype P53 (10, 11). Additionally, stabilization of P53 in the absence of detectable N-and C-terminal phosphorylation engenders full P53 activation (12). Thus, posttranslational P53 modifications fine-tune P53 transcription responses but do not act as on-off switches.A second model is supported by accumulating evidence that damage-activated kinases also phosphorylate Hdm2 to switch its E3 ligase specificity from P53 to itself and Hdmx (13-17). In part, the switch in substrate specificity is mediated by posttranslational modifications of Hdm2 and Hdmx that promote dissociation of the deubiquitinating enzyme HAUSP from Hdm2 and Hdmx (18). This increases Hdm2 and Hdmx degradation, resulting in P53 stabilization, accumulation, and transcriptional activation. Whether increasing Hdm2 levels is required for efficient Hdmx degradation after DNA damage remains unclear (13, 19). Together these data suggest a model in which destabilization and degradation of Hdm2 and Hdmx are critical for P53 stabilization and activation.Subtle perturbations in Hdm2 and Hdmx stoichiometry profoundly alter P53 activity and tumor sup...

Comparative analysis of mutants using transfection is complicated by clones exhibiting variable levels of gene expression due to copy number differences and genomic position effects. Recombinase-mediated cassette exchange (RMCE) can overcome these problems by introducing the target gene into pre-determined chromosomal loci, but recombination between the available recombinase targeting sites can reduce the efficiency of targeted integration. We developed a new LoxP site (designated L3), which when used with the original LoxP site (designated L2), allows highly efficient and directional replacement of chromosomal DNA with incoming DNA. A total of six independent LoxP integration sites introduced either by homologous recombination or retroviral delivery were analyzed; 70–80% of the clones analyzed in hamster and human cells were correct recombinants. We combined the RMCE strategy with a new, tightly regulated tetracycline induction system to produce a robust, highly reliable system for inducible transgene expression. We observed stable inducible expression for over 1 month, with uniform expression in the cell population and between clones derived from the same integration site. This system described should find significant applications for studies requiring high level and regulated transgene expression and for determining the effects of various stresses or oncogenic conditions in vivo and in vitro.

BH3 mimetic compounds induce tumor cell death through targeted inhibition of anti-apoptotic BCL2 proteins. Resistance to one such compound, ABT-737, is due to increased levels of anti-apoptotic MCL1. Using chemical and genetic approaches, we show that resistance to ABT-737 is abrogated by inhibition of the mitochondrial RING E3 ligase, MARCH5. Mechanistically, this is due to increased expression of pro-apoptotic BCL2 family member, NOXA, and is associated with MARCH5 regulation of MCL1 ubiquitylation and stability in a NOXA-dependent manner. MARCH5 expression contributed to an 8-gene signature that correlates with sensitivity to the preclinical BH3 mimetic, navitoclax. Furthermore, we observed a synthetic lethal interaction between MCL1 and MARCH5 in MCL1-dependent breast cancer cells. Our data uncover a novel level at which the BCL2 family is regulated; furthermore, they suggest targeting MARCH5-dependent signaling will be an effective strategy for treatment of BH3 mimetic-resistant tumors, even in the presence of high MCL1.

The yeast Sir2/3/4 protein complex forms a repressive heterochromatin-like structure that confers transcriptional silencing. The complex nucleates at silencers and then spreads distally by a process that requires the NAD+-dependent deacetylase activity of Sir2 and the affinity of Sir3/4 for deacetylated histone tails. A byproduct of the Sir2 reaction, O-acetyl-ADP-ribose (OAADPr), is thought to aid spreading by binding one of the Sir proteins. We have used a protein chimera approach to reexamine the essential roles of Sir2 in silencing. We show that a Sir3 chimera bearing Hos3, an unrelated NAD+-independent histone deacetylase, substitutes for Sir2 in transcriptional repression. Sir3-Hos3 requires silencers and Sir4, consistent with the chimera operating within the Sir pathway. Furthermore, the chimera silences in strains lacking all five OAADPr-producing deacetylases, indicating that the OAADPr is not required. Silencing by an analogous Hos3 hybrid bearing the Sir2 targeting motifs shows that the motifs operate independently of the Sir2 reaction. Together, these data demonstrate that protein deacetylation is the only necessary function of Sir2 in the creation of silenced chromatin.

Cell-based assays of protein-protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs). This is since split luciferase offers a greater signal:noise ratio and, unlike split FPs, the PPI can be reversed upon small molecule treatment. While multiplexed PPI assays using luciferase have been reported, they suffer from low signal:noise and require fairly complex spectral deconvolution during analysis. Furthermore, the luciferase enzymes used are large, which limits the range of PPIs that can be interrogated due to steric hindrance from the split luciferase fragments. Here, we report a multiplexed PPI assay based on split luciferases from Photinus pyralis (firefly luciferase, FLUC) and the deep-sea shrimp, Oplophorus gracilirostris (NanoLuc, NLUC). Specifically, we show that the binding of the p53 tumor suppressor to its two major negative regulators, MDM2 and MDM4, can be simultaneously measured within the same sample, without the requirement for complex filters or deconvolution. We provide chemical and genetic validation of this system using MDM2-targeted small molecules and mutagenesis, respectively. Combined with the superior signal:noise and smaller size of split NanoLuc, this multiplexed PPI assay format can be exploited to study the induction or disruption of pairwise interactions that are prominent in many cell signaling pathways.

Purpose: Tumor blood vessels exhibit morphological and functional aberrancies. Its maturity and functionality are closely associated with colon cancer progression and therapeutic efficacy. The direct evidence proving whether oridonin (ORI) has vascular normalization promoting effect from which combination therapies will benefit is still lacking. Methods: We established a subcutaneous xenograft model of human colon cancer. The animals were divided into the Control and ORI-treated groups. Immunohistochemical analysis and TUNEL staining was applied to evaluate the proliferation, apoptosis and angiogenesis. Western blot analysis was employed to characterize the angiogenesis-related factors and JAK2/STAT3 signaling. Then, vascular normalization and macrophage reprogramming were assessed by immunofluorescence analysis. Results: The results showed that ORI obviously reduced tumor growth, diminished the numbers of Ki67 + cells and CD31 + microvessel density, while increased the numbers of TUNEL + cells. The expression levels of VEGF and bFGF proteins were dramatically down-regulated while the angiostatin and endostatin levels were increased in the ORI-treated group. Moreover, ORI therapy remarkably promoted the pericyte coverage of tumor vessels from days 5 to 10, with the highest pericyte coverage rate occurred at day 7. In the time window of vascular normalization, hypoxia of the tumor microenvironment was improved by ORI, the expression of HIF-1a was downregulated. Moreover, CD206 + macrophage cells were diminished in the ORI-treated group. These anticancer effects of ORI maybe partly mediated by suppressing JAK2/STAT3 signaling pathway. Conclusions: These results highlight the potential effect of ORI on anti-angiogenesis and inducing vessel normalization roles of ORI, and probably provide optimum time point for the ORI therapy in conjunction with the chemoradiotherapy or immunotherapy.