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

Human Cerberus Prevents Nodal-Receptor Binding, Inhibits Nodal Signaling, and Suppresses Nodal-Mediated Phenotypes

<div><p>The Transforming Growth Factor-ß (TGFß) family ligand Nodal is an essential embryonic morphogen that is associated with progression of breast and other cancers. It has therefore been suggested that Nodal inhibitors could be used to treat breast cancers where Nodal plays a defined role. As secreted antagonists, such as Cerberus, tightly regulate Nodal signaling during embryonic development, we undertook to produce human Cerberus, characterize its biochemical activities, and determine its effect on human breast cancer cells. Using quantitative methods, we investigated the mechanism of Nodal signaling, we evaluated binding of human Cerberus to Nodal and other TGFß family ligands, and we characterized the mechanism of Nodal inhibition by Cerberus. Using cancer cell assays, we examined the ability of Cerberus to suppress aggressive breast cancer cell phenotypes. We found that human Cerberus binds Nodal with high affinity and specificity, blocks binding of Nodal to its signaling partners, and inhibits Nodal signaling. Moreover, we showed that Cerberus profoundly suppresses migration, invasion, and colony forming ability of Nodal expressing and Nodal supplemented breast cancer cells. Taken together, our studies provide mechanistic insights into Nodal signaling and Nodal inhibition with Cerberus and highlight the potential value of Cerberus as anti-Nodal therapeutic.</p></div

Equilibrium binding and rate constants.

<p>(est): Binding rates were calculated by separately fitting association and dissociation rate constants for each concentration and taking the average of the calculated binding rate constants.</p><p>†: Binding rates were calculated by fitting each individual concentration and taking the average of the calculated binding rate constants.</p><p>Equilibrium binding and rate constants.</p

Cerberus suppresses breast cancer cell colony-forming ability.

<p><b><i>(A)</i></b> Representative images of colony formation assay for MCF-7 (left) and MDA-MB-231 cells (right) (10X magnification). Cells were grown in serum containing medium supplemented with 0 nM (top, control), 17.8 nM (not shown) or 178 nM (bottom) Cerberus. <b><i>(B)</i></b> Analysis of colony formation assay for MCF-7 (right) and MDA-MB-231 (left) cells (3A). Images were analyzed using ImageJ to determine number of colonies. Experiments were carried out with 0 nM (blue), 17.8 nM (red), or 178 nM (green) Cerberus. Colony formation assays were performed in triplicates in 6 well plates.</p

Cerberus ligand binding.

<p><b><i>(A)</i></b> Recombinant Cerberus construct. Full length human Cerberus was fused at the C-terminus to a human Igg1 Fc fragment via a linker containing a TEV cleavage site. <b><i>(B)</i></b> Coomassie Blue stained SDS-PAGE of Cerberus shown on the left side of the panel, Western blot using anti-Cerberus antibody RnD Systems, AF1075 is shown on the right side of the panel. Recombinant Cerberus is purified from Chinese Hamster Ovary cell conditioned medium using protein A capture. Overall, Cerberus-Fc is pure; size heterogeneity may be introduced by variations in glycan structure. The observed smaller Cerberus-Fc fragment could correspond is likely a proteolytic product. We have tested a Cerberus construct that is smaller than the proteolytic product (manuscript in preparation). Nodal binding activity of the shorter Cerberus-Fc is indistinguishable from full-length Cerberus-Fc. <b><i>(C)</i></b> Nodal-Cerberus interaction. Cerberus-Fc was immobilized on an SPR sensor chip and different concentrations of Nodal were injected as shown. The Nodal-Cerberus association constant (<i>k_a</i>) is 1.3×10⁴ M^-1s^-1, the dissociation constant (<i>k_d</i>) is 1.4×10^-5 s^-1, and the equilibrium dissociation constant (<i>K_d</i>) is 1.0 nM. Fitted curves (orange lines) are superimposed over experimental curves. <b><i>(D)</i></b> BMP-2-Cerberus interaction. Cerberus-Fc was immobilized on an SPR sensor chip and different concentrations of BMP-2 were injected as shown. The sensograms could not be fitted to a global kinetic model due to the extremely fast dissociation rate. Single curve fitting and averaging yielded an estimated BMP-2-Cerberus association rate constant (<i>k_a</i>) of ~2.4×10⁴ M^-1s^-1, a dissociation constant (<i>k_d</i>) of ~0.072 s^-1, and an equilibrium dissociation constant (<i>K_d</i>) of ~3,000 nM. <b><i>(E)</i></b> GDF-11-Cerberus interaction. Cerberus-Fc was immobilized on an SPR sensor chip and different concentrations of GDF-11 were injected as shown. The sensograms were fit to a global kinetic model that yielded an association rate constant of 1.2×10³ M^-1s^-1, a dissociation rate constant of 0.014 s^-1, and an equilibrium dissociation constant of 5,800 nM. Fitted curves (grey lines) are superimposed over experimental curves. <b><i>(F)</i></b> Comparison of ligand binding to human Cerberus. Cerberus-Fc was immobilized on an SPR sensor chip and different TGFß family ligands were injected at a concentration of 80 nM as shown. Injections were performed at 40 µl/min. Ligands marked with an asterisk (*), including Nodal, BMP2 and others (Activin A and BMP4), have been shown to interact with Cerberus of different species. Nodal (red) most convincingly binds human Cerberus.</p

Nodal-receptor interactions.

<p><b><i>(A)</i></b> Nodal binding to ACTRIIB. ACTRIIB-Fc was immobilized on an SPR sensor chip and different concentrations of Nodal were injected as shown. Green vertical bar corresponds to begin of analyte (Nodal) injection (association); the blue vertical bar corresponds to begin of dissociation. The ACTRIIB-Fc-Nodal sensograms could not be fitted to a global kinetic model. Instead, an equilibrium dissociation constant (<i>K_d</i>) of 10 nM was estimated by separately fitting association and dissociation curves. <b><i>(B)</i></b> Nodal binding to ACTRIIA. ACTRIIA-Fc was immobilized on an SPR sensor chip and different concentrations of Nodal were injected as shown. The sensograms fitted a global kinetic model well, giving an association rate constant (<i>k_a</i>) of 2.0×10⁴ M^-1s^-1, a dissociation rate constant (<i>k_d</i>) of 2.0×10^-3 s^-1, and an equilibrium dissociation constant (<i>K_d</i>) of 100 nM. Fitted curves (orange lines) are superimposed over experimental curves. <b><i>(C)</i></b> Nodal binding to ALK4. ALK4-Fc was immobilized on an SPR sensor chip and different concentrations of Nodal were injected as shown. Like the Nodal-ACTRIIB interaction, Nodal-ALK4 sensograms could not be fitted to a global kinetic model. Instead, an equilibrium dissociation constant (<i>K_d</i>) of 15 nM was estimated by separately fitting association and dissociation curves. <b><i>(D)</i></b> Activin A binding to ALK4. ALK4-Fc was immobilized on an SPR sensor chip and different concentrations of Activin A were injected as shown. The sensograms fitted a global kinetic model very well, giving an association rate constant of 2.0×10⁵ M^-1s^-1, a dissociation rate constant of 4.8×10^-4 s^-1, and an equilibrium dissociation constant of 2.4 nM. Fitted curves (orange lines) are superimposed over experimental curves. (<i>E)</i> Nodal binding to BMPRII-Fc. BMPRII-Fc was immobilized on an SPR sensor chip and different concentrations of Nodal were injected as shown. Like the Activin A-ALK4 sensogram, the Nodal-BMRPII sensograms fitted a global kinetic model very well, giving an association rate constant of 3.1×10⁵ M^-1s^-1, a dissociation rate constant of 4.6×10^-5 s^-1, and an equilibrium dissociation constant of 0.149 nM. Fitted curves (orange lines) are superimposed over experimental curves. <b><i>(F)</i></b> Nodal binding to Cripto-1-Fc. Cripto-1-Fc was immobilized on an SPR sensor chip and different concentrations of Nodal were injected as shown. The Nodal-Cripto-1 sensograms fitted a global kinetic model, giving an association rate constant of 1.0×10⁴ M^-1s^-1, a dissociation rate constant of 2.6×10^-4 s^-1, and an equilibrium dissociation constant of 16 nM. Fitted curves (orange lines) are superimposed over experimental curves. <b><i>(G)</i></b> Nodal binding to Cryptic-Fc. Cryptic-Fc was immobilized on an SPR sensor chip and various concentrations of Nodal were injected as shown. The Nodal-Cryptic sensograms were fitted individually and an equilibrium dissociation constant (<i>K_d</i>) of 2,000 nM was calculated by taking the average of association and dissociation rate constants for each binding curve, giving an average association rate constant (<i>k_a</i>) of 5.5×10² M^-1s^-1 and an average dissociation rate constant (<i>k_d</i>) of 1.0×10^-3 s^-1. Individually fitted curves cannot be displayed. <b><i>(H)</i></b> Comparison of Nodal binding to Cripto-1, Cryptic, ALK4 and ALK7. Equal amounts of Fc fusion proteins as determined by SPR response units were immobilized on the SPR sensor chip and 80 nM Nodal was injected. Cripto-1 (red) shows the best binding, ALK4 and Cryptic (blue and green, respectively) bind Nodal with similar profiles. ALK7 (purple) obtained from RnD Systems and reconstituted as suggested does not bind Nodal.</p

Sequence comparison between TGFß family proteins of different species.

<p>Mature ligand and receptor/co-receptor ecto domains were compared. Identities were calculated relative to the human proteins over the entire conserved region.</p><p>Sequence comparison between TGFß family proteins of different species.</p

Cerberus inhibits breast cancer cell migration.

<p><b><i>(A)</i></b> Cerberus prevents wound closure of Nodal expressing breast cancer cells. MCF-7, Hs578t, BT-549 and MDA-MB-231 cells were plated in Ibidi culture insert dishes. Cells were grown in complete medium to 80% confluence, inserts were removed to create a gap and medium was replaced with complete medium supplemented with 2.5 μg/ml Mitomycin C and 0 nM (left panel), 17.8 nM (middle panel), or 178 nM (right panel) Cerberus. Images were taken at 0 h and 24 h after removing insert. <b><i>(B)</i></b> Wound closure evaluation. Images taken at 0 h (blue) and 24 h (green) were analyzed using Wimasis software (Ibidi) to quantify cell migration. Graphs within a panel correspond to experiments carried out with 0 nM (left), 17.8 nM (middle), or 178 nM (right) Cerberus.</p

Nodal induces breast cancer cell proliferation and invasion.

<p><b><i>(A)</i></b> Nodal induced cell proliferation. T47D breast cancer cells were grown for 48 h with or without Nodal (39 nM) and/or Cerberus (178 nM). Viable cell number was determined at 0 h (blue), 24 h (red), and 48 h (green) by measuring cellular ATP. <b><i>(B)</i></b> Breast cancer cell proliferation. MCF-7, Hs578t, BT-549, and MDA-MB-231, were grown in the presence of 0 nM (blue), 17.8 nM (red), or 178 nM (green) Cerberus. Viable cell number was determined at 0 h (left), 24 h (middle), and 48 h (right) following addition of Cerberus. <b><i>(C)</i></b> Nodal induced cell invasion. T47D cells were grown for 24 h in a Boyden Chamber. Growth medium contained 2.5 μg/ml Mitomycin C, 0 nM (left) or 39 nM (right) Nodal and 0 nM (blue), 17.8 nM (red), or 178 nM (green) Cerberus. A Cultrex Basement Membrane Extract (BME) coated filter separated the upper and lower chambers. Cell invasion was quantified using Calcein-AM fluorescence. <b><i>(D)</i></b> Cerberus inhibits invasion of Nodal expressing human breast cancer cells. MCF-7, Hs578t, BT-549, and MDA-MB-231, were placed in the top well of Boyden chamber in serum free medium containing 0 nM (blue), 17.8 nM (red) or 178 nM (green) Cerberus and 2.5 μg/ml Mitomycin C. The bottom chamber was filled with matching medium supplemented with serum. The filter separating top and bottom chambers was coated with BME. Cell invasion was quantified using Calcein-AM fluorescence.</p