Myc Cooperates with Ras by Programming Inflammation and Immune Suppression.

The two oncogenes KRas and Myc cooperate to drive tumorigenesis, but the mechanism underlying this remains unclear. In a mouse lung model of KRasG12D-driven adenomas, we find that co-activation of Myc drives the immediate transition to highly proliferative and invasive adenocarcinomas marked by highly inflammatory, angiogenic, and immune-suppressed stroma. We identify epithelial-derived signaling molecules CCL9 and IL-23 as the principal instructing signals for stromal reprogramming. CCL9 mediates recruitment of macrophages, angiogenesis, and PD-L1-dependent expulsion of T and B cells. IL-23 orchestrates exclusion of adaptive T and B cells and innate immune NK cells. Co-blockade of both CCL9 and IL-23 abrogates Myc-induced tumor progression. Subsequent deactivation of Myc in established adenocarcinomas triggers immediate reversal of all stromal changes and tumor regression, which are independent of CD4+CD8+ T cells but substantially dependent on returning NK cells. We show that Myc extensively programs an immune suppressive stroma that is obligatory for tumor progression

Tandem E2F Binding Sites in the Promoter of the p107 Cell Cycle Regulator Control p107 Expression and Its Cellular Functions

The retinoblastoma tumor suppressor (Rb) is a potent and ubiquitously expressed cell cycle regulator, but patients with a germline Rb mutation develop a very specific tumor spectrum. This surprising observation raises the possibility that mechanisms that compensate for loss of Rb function are present or activated in many cell types. In particular, p107, a protein related to Rb, has been shown to functionally overlap for loss of Rb in several cellular contexts. To investigate the mechanisms underlying this functional redundancy between Rb and p107 in vivo, we used gene targeting in embryonic stem cells to engineer point mutations in two consensus E2F binding sites in the endogenous p107 promoter. Analysis of normal and mutant cells by gene expression and chromatin immunoprecipitation assays showed that members of the Rb and E2F families directly bound these two sites. Furthermore, we found that these two E2F sites controlled both the repression of p107 in quiescent cells and also its activation in cycling cells, as well as in Rb mutant cells. Cell cycle assays further indicated that activation of p107 transcription during S phase through the two E2F binding sites was critical for controlled cell cycle progression, uncovering a specific role for p107 to slow proliferation in mammalian cells. Direct transcriptional repression of p107 by Rb and E2F family members provides a molecular mechanism for a critical negative feedback loop during cell cycle progression and tumorigenesis. These experiments also suggest novel therapeutic strategies to increase the p107 levels in tumor cells

Reactivation of Myc transcription in the mouse heart unlocks its proliferative capacity

Abstract: It is unclear why some tissues are refractory to the mitogenic effects of the oncogene Myc. Here we show that Myc activation induces rapid transcriptional responses followed by proliferation in some, but not all, organs. Despite such disparities in proliferative response, Myc is bound to DNA at open elements in responsive (liver) and non-responsive (heart) tissues, but fails to induce a robust transcriptional and proliferative response in the heart. Using heart as an exemplar of a non-responsive tissue, we show that Myc-driven transcription is re-engaged in mature cardiomyocytes by elevating levels of the positive transcription elongation factor (P-TEFb), instating a large proliferative response. Hence, P-TEFb activity is a key limiting determinant of whether the heart is permissive for Myc transcriptional activation. These data provide a greater understanding of how Myc transcriptional activity is determined and indicate modification of P-TEFb levels could be utilised to drive regeneration of adult cardiomyocytes for the treatment of heart myopathies

Fundulus as the premier teleost model in environmental biology : opportunities for new insights using genomics

Author Posting. © Elsevier B.V., 2007. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Comparative Biochemistry and Physiology Part D: Genomics and Proteomics 2 (2007): 257-286, doi:10.1016/j.cbd.2007.09.001.A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.This material is based on work supported by grants from the National Science Foundation DBI-0420504 (LJB), OCE 0308777 (DLC, RNW, BBR), BES-0553523 (AW), IBN 0236494 (BBR), IOB-0519579 (DHE), IOB-0543860 (DWT), FSML-0533189 (SC); National Institute of Health NIEHS P42-ES007381(GVC, MEH), P42-ES10356 (RTD), ES011588 (MFO); and NCRR P20 RR-016463 (DWT); Natural Sciences and Engineering Research Council of Canada Discovery (DLM, TDS, WSM) and Collaborative Research and Development Programs (DLM); NOAA/National Sea Grant NA86RG0052 (LJB), NA16RG2273 (SIK, MEH,GVC, JJS); Environmental Protection Agency U91620701 (WSB), R82902201(SC) and EPA’s Office of Research and Development (DEN)

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

E2F binding sites mediate activation of the <i>p107</i> promoter in cycling cells.

<p>(A) RT-qPCR analysis of <i>p107</i> mRNA relative to <i>TBP</i> in asynchronously cycling primary wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEFs. (n = 12) (B) Immunoblot analysis (left panel) of p107 expression in wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEFs as in A. Tubulin expression is shown as a loading control. p107 protein quantification (right panel) is shown relative to Tubulin levels. (n = 3) (C) Representative example of Hoescht33342 staining of asynchronously cycling MEFs showing G1 and S phase populations; wild-type and mutant cells have similar profiles (data not shown). (D) RT-qPCR analysis of immortalized WT, <i>p107^E2F-1*/1*</i> and <i>p107^{E2F-1*2*/1*2*}</i> MEFs. For each genotype, G0 samples were collected after at least three days of serum starvation. Asynchronous cells were stained with Hoechst33342 and sorted by their DNA content into G1 and S-phase samples. (n≥2) (E) and (F) RT-qPCR analysis of primary wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEFs that have been synchronized in G0 by serum starvation. DMEM supplemented with 20% serum was added at time 0, and extracts were collected at 10 hrs, 16 hrs, 22 hrs, and 28 hrs post-stimulation. (E) <i>p107</i> mRNA and (F) <i>Cdc6</i> mRNA. n≥8 for both genotypes at all time points. (G) Percentage of cells in S-phase in primary MEFs collected during cell-cycle re-entry as in E. and F. Percentages were calculated by BrdU/PI analysis (n = 3). (H) Immunoblot analysis of p107 protein expression in primary wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEF extracts collected at 0 hr, 8 hrs, 12 hrs, 16 hrs, 20 hrs, and 24 hrs post-stimulation with 20% serum. MCM6 expression is shown as a positive control for cell cycle re-entry, and Tubulin levels are shown as a loading control. Note that the second, slowly migrating form of p107 at later time points probably reflects p107 phosphorylation during S phase.</p

Regulation of the mouse <i>p107</i> promoter through E2F binding sites in reporter assays.

<p>(A) Conservation of the proximal <i>p107</i> promoter across mammalian species. The two tandem consensus E2F binding sites (BS1 and BS2) are each indicated by a box. (B) Schematic representation of wild-type (WT), p107-1*, p107-2*, and p107-1*2* luciferase vectors. Transcription factor binding sites contained in this promoter region, as identified by sequence analysis, are indicated, as is the transcription start site (arrow). Black rectangular boxes indicate E2F consensus sites; white boxes indicate E2F consensus sites that are mutated. The inset represents the mutations (aaa) introduced in each site. (C) Relative luciferase activity expressed by the four constructs, co-transfected with CMV-E2F3 (+) or empty pCDNA (−), in cycling mESCs. For statistical analysis, each mutant construct was compared to the wild-type one and the effect of E2F3 on each construct was analyzed. (n = 3) (D) Relative luciferase activity in quiescent MEFs. (n = 15) (E) Comparison of the models for the regulation of the human and mouse <i>p107</i> promoters by E2F based on reporter assays. Gradient triangles indicate the relative importance of each consensus E2F site to either activation or repression of <i>p107</i>.</p

Altered <i>p107</i> expression affects cellular proliferation.

<p>(A,B) Immortalized wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEFs were synchronized in G0 through at least three days of serum starvation. DMEM supplemented with 20% BGS was used to stimulate cell-cycle entry. Extracts were collected at the number of hours indicated post-serum stimulation. (A) RT-qPCR analysis of <i>Cdc6</i> mRNA in wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEFs. (n = 3) (B) Percentage of cells in S-phase, as determined by BrdU/PI staining, at the indicated time points. (n≥4) (C) Cell-cycle profiles of asynchronous primary wild-type, <i>p107^{E2F-1*2*/1*2*}</i>, and <i>p107^−/−</i> MEFs. Percentages of cells in each phase were determined by BrdU/PI staining. (n≥2) (D) Cellular proliferation of primary wild-type, <i>p107^{E2F-1*2*/1*2*}</i>, and <i>p107^−/−</i> MEFs. Equal numbers of cells were plated at day 0. Cells were then counted every other day from day 1 to day 9 post-plating. For statistical analysis, <i>p107^{E2F-1*2*/1*2*}</i> cells were compared to wild-type cells at each time point. (n≥13) (E) Model for the context-dependent regulation of <i>p107</i> transcription by E2F family members. In cycling mESCs, activating members of the E2F family such as E2F3 bind to the <i>p107</i> promoter mostly through the distal consensus E2F binding site (site 1). In quiescent MEFs, binding of the E2F4 repressor is also largely dependent on the presence of the distal consensus site. However, E2F4 may also be recruited to the <i>p107</i> promoter through interactions with other transcription factors and/or by binding to other DNA sequences. The size of the E2F boxes indicates the relative binding activity.</p