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

Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

<p>Abstract</p> <p>Background</p> <p>Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs (Rb-/- MEFs).</p> <p>Findings</p> <p>Comparative analysis of the expression profiles of 3T3-L1 cells and wild-type MEFs revealed genes involved specifically in early regulation of the adipocyte differentiation as well as secreted factors and signaling molecules regulating the later phase of differentiation. In an attempt to identify transcription factors regulating adipogenesis, bioinformatics analysis of the promoters of coordinately and highly expressed genes was performed. We were able to identify a number of high-confidence target genes for follow-up experimental studies. Additionally, combination of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1.</p> <p>To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis of 64 deregulated genes showed that the Rb-/- MEF model exhibits a brown(-like) adipocyte phenotype. Additionally, the analysis results indicate a different or additional role for pRb family member involvement in the lineage commitment.</p> <p>Conclusion</p> <p>In this study a number of commonly modulated genes during adipogenesis in 3T3-L1 cells and MEFs, potential transcriptional regulation mechanisms, and differentially regulated targets during adipocyte differentiation of Rb-/- MEFs could be identified. These data and the analysis provide a starting point for further experimental studies to identify target genes for pharmacological intervention and ultimately remodeling of white adipose tissue into brown adipose tissue.</p

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

CMB-S4

We describe the stage 4 cosmic microwave background ground-based experiment CMB-S4

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

<i>p107</i> repression in quiescent MEFs is mediated by the two E2F binding sites.

<p>(A) mESCs targeted by the neomycin resistance cassette but retaining a wild-type <i>p107</i> promoter and mESCs targeted by homozygous mutations into the distal (1*/1*) or both E2F sites (1*2*/1*2*) were injected to generate chimeric embryos. Wild-type, <i>p107^E2F-1*/1*</i> and <i>p107^{E2F-1*2*/1*2*}</i> MEFs derived from chimeric embryos were selected for Neomycin resistance to generate pure populations. (B) RT-qPCR analysis of <i>p107</i> expression in quiescent wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEFs. (n≥9) (C) Immunoblot analysis of p107 in the same conditions. Tubulin expression is shown as a loading control. (D) Quantitative ChIP analysis of E2F4, p107, and p130 binding on the <i>p107</i> promoter in quiescent immortalized wild-type, <i>p107^E2F-1*/1*</i>, and <i>p107^{E2F-1*2*/1*2*}</i> MEFs. The <i>B-Myb</i> promoter is shown as a control. (n = 3) (E) Quantitative ChIP analysis of Rb binding to the <i>p107</i> and <i>Mcm3</i> promoters in cycling immortalized wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEFs. Mouse IgG antibodies serve as a negative control. (n≥3) For (D,E), fold enrichment is calculated over <i>actin</i> and the y-axis is plotted on a <i>log2</i> scale.</p

CD11c-expressing Ly6C+CCR2+ monocytes constitute a reservoir for efficient Leishmania proliferation and cell-to-cell transmission.

The virulence of intracellular pathogens such as Leishmania major (L. major) relies largely on their ability to undergo cycles of replication within phagocytes, release, and uptake into new host cells. While all these steps are critical for successful establishment of infection, neither the cellular niche of efficient proliferation, nor the spread to new host cells have been characterized in vivo. Here, using a biosensor for measuring pathogen proliferation in the living tissue, we found that monocyte-derived Ly6C+CCR2+ phagocytes expressing CD11c constituted the main cell type harboring rapidly proliferating L. major in the ongoing infection. Synchronization of host cell recruitment and intravital 2-photon imaging showed that these high proliferating parasites preferentially underwent cell-to-cell spread. However, newly recruited host cells were infected irrespectively of their cell type or maturation state. We propose that among these cells, CD11c-expressing monocytes are most permissive for pathogen proliferation, and thus mainly fuel the cycle of intracellular proliferation and cell-to-cell transfer during the acute infection. Thus, besides the well-described function for priming and activating T cell effector functions against L. major, CD11c-expressing monocyte-derived cells provide a reservoir for rapidly proliferating parasites that disseminate at the site of infection