Structural and functional analyses of nucleosomes containing histone variant H2A.Z have drawn a lot of interest over the past few years. Important work in budding yeast has shown that H2A.Z (Htz1)-containing nucleosomes are specifically located on the promoter regions of genes, creating a specific chromatin structure that is poised for disassembly during transcription activation. The SWR1 complex is responsible for incorporation of Htz1 into nucleosomes through ATP-dependent exchange of canonical H2A-H2B dimers for Htz1-H2B dimers. Interestingly, the yeast SWR1 complex is functionally linked to the NuA4 acetyltransferase complex in vivo. NuA4 and SWR1 are physically associated in higher eukaryotes as they are homologous to the TIP60/p400 complex, which encompasses both histone acetyltransferase (Tip60) and histone exchange (p400/Domino) activities. Here we present work investigating the impact of NuA4-dependent acetylation on SWR1-driven incorporation of H2A.Z into chromatin. Using in vitro histone exchange assays with native chromatin, we demonstrate that prior chromatin acetylation by NuA4 greatly stimulates the exchange of H2A for H2A.Z. Interestingly, we find that acetylation of H2A or H4 N-terminal tails by NuA4 can independently stimulate SWR1 activity. Accordingly, we demonstrate that mutations of H4 or H2A N-terminal lysine residues have similar effects on H2A.Z incorporation in vivo, and cells carrying mutations in both tails are nonviable. Finally, depletion experiments indicate that the bromodomain-containing protein Bdf1 is important for NuA4-dependent stimulation of SWR1. These results provide important mechanistic insight into the functional cross-talk between chromatin acetylation and ATP-dependent exchange of histone H2A variants.Genetic information within the eukaryotic cell nucleus is organized in a highly conserved structural polymer, chromatin, which supports and controls crucial functions of the genome. Chromatin remodeling and post-translational modifications of histones are critical processes regulating genome expression and maintenance by controlling access to DNA and signaling local regions for specific molecular interactions (1, 2). Incorporation of different histone variants in specific chromosomal regions is also known to be associated with important biological processes controlling gene expression and genome integrity (3).A specific histone variant, H2A.Z, has been the focus of intense research over the past few years (4 -6). Delineation of its impact on nucleosome structure and stability and on gene expression has turned into a long-heated debate involving apparently contradicting experimental data. However, recent findings led to an emerging model that could partly reconcile these contradictions (7). Genome-wide analyses of H2A.Z localization in eukaryotes indicate that it is preferentially found on gene promoter/regulatory regions within nucleosomes flanking more accessible DNA sequences (8 -14). Importantly, H2A.Z is found within the promoters of inactive or weakly transcribed ge...

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for infections that cause a substantial amount of death, suffering, and loss around the world. Still, relatively little is known about the mechanisms of gene expression in these bacteria. Here, we used genome-wide location assays to identify direct target genes for mycobacterial factors. Chromatin immunoprecipitation assays were performed with M. bovis BCG for Myc-tagged proteins expressed using an anhydrotetracycline-inducible promoter, and enriched DNA fragments were hybridized to a microarray representing intergenic regions from the M. tuberculosis H37Rv genome. Several putative target genes were validated by quantitative PCR. The corresponding transcriptional start sites were identified for F , C , and K , and consensus promoter sequences are proposed. Our conclusions were supported by the results of in vitro transcription assays. We also examined the role of each holoenzyme in the expression of factor genes. Our results revealed that many factors are expressed from autoregulated promoters.Several pathogenic mycobacteria cause infections that lead to tuberculosis or related diseases in a variety of organisms. For example, Mycobacterium tuberculosis infects nearly onethird of the human population (10), while Mycobacterium bovis infections in cattle are responsible for important agricultural losses (12). Advanced knowledge of the biology of these bacteria could significantly contribute to prevention and treatment of the associated diseases. A key step toward this end occurred with the publication of the complete M. tuberculosis and M. bovis genome sequences (8, 12). Several genes have also been interrupted in M. tuberculosis, leading to different outcomes for virulence (for reviews, see references 7 and 38). More recently, in many studies workers have reported gene expression profiles for various growth conditions and during different stages of infection in mice and human macrophages (21, 37, 41), providing new insights into the strategies used by the pathogens to adapt to various environments. Nevertheless, much remains to be discovered about the molecular mechanisms controlling the genetic programs in pathogenic mycobacteria.Thirteen factor genes were predicted based on the genome sequence of M. tuberculosis, and almost identical M. bovis counterparts were also predicted (25,35). Some of these genes may orchestrate critical responses in the pathogenesis of tuberculosis and therefore may be attractive targets for development of new drugs and vaccines. Indeed, in mouse models infections with sigC, sigD, sigE, sigF, sigh, and sigL mutant strains were all attenuated compared to infections with the wild-type strains (5,9,13,16,20,23,24,26,27,32,33,40). Still, the conditions leading to the activities of most factors remain elusive (23,35). Differential gene expression profiles were nonetheless obtained for the factor mutant strains, and consensus promoter sequences have been proposed (5,9,13,16,20,26,27,32,33,40). However, direct and indirect effec...

Cloning and transplantation of bacterial genomes is a powerful method for the creation of engineered microorganisms. However, much remains to be understood about the molecular mechanisms and limitations of this approach. We report the whole-genome cloning of Mesoplasma florum in Saccharomyces cerevisiae, and use this model to investigate the impact of a bacterial chromosome in yeast cells. Our results indicate that the cloned M. florum genome is subjected to weak transcriptional activity, and causes no significant impact on yeast growth. We also report that the M. florum genome can be transplanted into Mycoplasma capricolum without any negative impact from the putative restriction enzyme encoding gene mfl307. Using whole-genome sequencing, we observed that a small number of mutations appeared in all M. florum transplants. Mutations also arose, albeit at a lower frequency, when the M. capricolum genome was transplanted into M. capricolum recipient cells. These observations suggest that genome transplantation is mutagenic, and that this phenomenon is magnified by the use of genome donor and recipient cell belonging to different species. No difference in efficiency was detected after three successive rounds of genome transplantation, suggesting that the observed mutations were not selected during the procedure. Taken together, our results provide a more accurate picture of the events taking place during bacterial genome cloning and transplantation.

Mesoplasma florum, a fast-growing near-minimal organism, is a compelling model to explore rational genome designs. Using sequence and structural homology, the set of metabolic functions its genome encodes was identified, allowing the reconstruction of a metabolic network representing~30% of its protein-coding genes. Growth medium simplification enabled substrate uptake and product secretion rate quantification which, along with experimental biomass composition, were integrated as species-specific constraints to produce the functional iJL208 genome-scale model (GEM) of metabolism. Genome-wide expression and essentiality datasets as well as growth data on various carbohydrates were used to validate and refine iJL208. Discrepancies between model predictions and observations were mechanistically explained using protein structures and network analysis. iJL208 was also used to propose an in silico reduced genome. Comparing this prediction to the minimal cell JCVI-syn3.0 and its parent JCVI-syn1.0 revealed key features of a minimal gene set. iJL208 is a stepping-stone toward model-driven whole-genome engineering.

Colon cancer is the second most common cause of cancer mortality in the Western world with metastasis commonly present at the time of diagnosis. Screening for propagation and metastatic behavior in a novel chimeric-mouse colon cancer model, driven by mutant p53 and β-Catenin, led to the identification of a unique, invasive adenocarcinoma. Comparison of the genome of this tumor, CB42, with genomes from non-propagating tumors by array CGH and sequencing revealed an amplicon on chromosome five containing CDK6 and CDK14, and a KRAS mutation, respectively. Single agent small molecule inhibition of either CDK6 or MEK, a kinase downstream of KRAS, led to tumor growth inhibition in vivo whereas combination therapy not only led to regression of the subcutaneous tumors, but also near complete inhibition of lung metastasis; thus, genomic analysis of this tumor led to effective, individualized treatment.

AVEO has developed a series of inducible mouse models of cancer which, through the preservation of critical tumor/stromal interactions, facilitate identification of cell-surface and secreted proteins that represent viable targets for therapeutic antibodies and other biologics. Functional genetic screens performed in vivo in these models identified the Notch pathway as a critical regulator of tumor maintenance. This finding is consistent with emerging evidence that activation of Notch signaling via receptor point mutation, receptor amplification, and elevated receptor and ligand expression, plays a key role in various human cancers. Moreover, the Notch pathway controls diverse aspects of tumorigenesis and tumor maintenance, regulating tumor autonomous processes and interactions with the microenvironment, including angiogenesis. To further understand the role of the Notch pathway in tumor maintenance, and to assess the therapeutic potential of targeting the Notch pathway in cancer, we have generated monoclonal antibodies that inhibit various Notch receptors. Characterization of a Notch1-specific monoclonal antibody through cell-based and biochemical studies demonstrated that the antibody bound to the Notch1 ligand binding domain with high affinity, prevented ligand mediated activation of the receptor, and specifically repressed Notch1-dependent signaling with high potency. Mice treated with the Notch1 antibody exhibited altered T cell fate specification and loss of hair pigmentation, as expected for loss of Notch1 function. Significantly, specific inhibition of Notch1 by this antibody did not result in the dose-limiting gut toxicity observed with pan-Notch inhibitors such as gamma-secretase inhibitors. However, strong inhibition of functional angiogenesis was observed upon antibody treatment in both in vitro and in vivo models. The apparent lack of toxicity of this antibody in mouse models suggests that inhibition of Notch1 could be effectively combined with other therapies to enhance anti-angiogenic effects, or to overcome resistance to VEGF/VEGFR inhibition. To identify tumors that are dependent upon tumor autonomous Notch signaling the expression of Notch pathway related genes was correlated with Notch pathway dependence in human cancer cell lines. Active Notch signaling alone did not predict dependence upon Notch, but expression of a single Notch target gene, HeyL, was highly correlated with sensitivity of human cancer cell lines to inhibition of ligand-dependent Notch signal. The utility of this biomarker was further confirmed by the identification of a subset of Kras mutant pancreatic and colon cancer cell lines that were subsequently demonstrated to be highly sensitive to Notch pathway inhibition. Hence, HeyL expression may serve as a predictive biomarker of Notch-dependent tumors. These data support the clinical development of AVEO's humanized Notch antibodies for the treatment of human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5170.

All solid tumors are thought to require neo-vascularization. Therefore pharmacologic inhibition of angiogenesis may represent an important component for treating many therapeutically challenging tumor types. One attractive anti-angiogenesis agent is the ATP competitive small molecule VEGFR inhibitor tivozanib (AV-951). Tivozanib exhibits picomolar inhibitory activity against all three VEGF receptors, a multi-day T1/2 in humans, and demonstrates robust clinical activity in renal cell carcinoma, the signal tumor type for VEGF pathway inhibition. To test preclinical efficacy of tivozanib in other cancer types, we chose primary mouse tumor models due to their ability to capture the complex heterotypic interactions between tumor cells and the microenvironments. Our mouse tumor model strategy involves stepwise genetic manipulation of embryonic stem (ES) cells and chimera formation to enable direct tumor induction in tissues containing both normal and engineered cells. A HER2 driven breast model as well as an allelic series of lung cancer models containing EGFR, KRAS, or HER2 oncogenes demonstrated that resultant adenocarcinomas arose within surrounding normal tissue and exhibited features of advanced malignancies. In this study, we tested the response of HER2 driven breast tumors as well as KRAS and EGFR driven lung tumors to the VEGFR inhibitor tivozanib in tumor bearing chimeric mice. We observed that although tivozanib treatment conferred significant survival benefit to the tumor bearing mice in all three models, it is not able to eradicate all tumor cells. Response to tivozanib in the breast model is much more heterogeneous than in the lung models, with some tumors exhibiting significant regression while others showing progression on treatment, either initially or after a period of response. In contrast, lung tumors showed more uniform response to tivozanib treatment initially, but tumors quickly grew back upon discontinuation of tivozanib treatment. These results indicate that both breast and lung cancer patients could potentially benefit from sustained anti-angiogenesis therapy, and combination of anti-angiogenesis with tumor targeting agents may be required for enduring efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1378.

Lung cancer is one of the most prevalent and deadly malignancies in the world, with more than 213,000 new cases diagnosed each year in the US and a five year survival rate of 15%. Activating mutations in a number of key signaling genes including KRAS, EGFR, and HER2 have been identified. Mutations in EGFR are highly correlated with sensitivity to small molecule EGFR inhibitors, although resistance tends to develop within a short period of time after initiation of treatment. Targeted inhibition of KRAS has not been successful so far, which in turn fueled efforts to identify tractable alternative therapeutic points of intervention. Because all solid tumors are thought to require neo-vascularization, pharmacologic angiogenesis inhibition may represent an attractive therapeutic strategy for these challenging tumor settings. One attractive anti-angiogenesis agent is the ATP competitive small molecule VEGFR inhibitor tivozanib (AV-951). tivozanib exhibits picomolar inhibitory activity against all three VEGF receptors, a multi-day T1/2 in humans, and demonstrates robust clinical activity demonstrated in renal cell carcinoma, the signal tumor type for VEGF pathway inhibition. In an effort to recapitulate the stochastic nature of human cancer in genetically engineered mouse models and to maximize their use in preclinical settings, we developed a mouse tumor model strategy involving stepwise genetic manipulation of embryonic stem (ES) cells and chimera formation to enable direct tumor induction in tissues containing both normal and engineered cells. An allelic series of lung cancer models containing EGFR, KRAS, or HER2 oncogenes demonstrated that resultant adenocarcinomas arising within normal lung tissue exhibited features of advanced malignancies. In this study, we tested the response of KRAS and EGFR driven lung tumors to the VEGFR inhibitor tivozanib in tumor bearing chimeric mice identified by bioluminescent imaging. We demonstrated that tivozanib treatment conferred significant survival benefit to the tumor bearing mice. Consistent with previous reports that tumors become more dependent on neo-angiogenesis for survival and proliferation, we observed sensitivity to tivozanib in advanced adenocarcinomas but not hyperplasia or small adenomas. In addition, we found that upon discontinuation of tivozanib treatment, tumors quickly grew back and became less sensitive to further tivozanib treatment. These results indicate that lung cancer patients could potential benefit from anti-angiogenesis therapy, but sustained inhibition of all VEGF receptors may be important to prevent development of resistance. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A22.