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C-myc/anti-myc Ab Interaction with Fusion Proteins

C-myc/anti-myc Ab Interaction with Fusion Proteins


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I am going to prepare a c-myc fusion protein with the following configuration:

(28-residue signal sequence)-(c-myc)-(GGSGGGSG Linker)-(Protein of Interest (POI))

POI is a transmembrane protein and the 28-residue signal sequence is needed for membrane incorporation in ER.

My concern is the disruption of the c-myc/anti-myc Ab interaction due to the fact that c-myc being fused on both ends. Does anyone have any experience or reference with a similar construct?


After some further research, I found papers reporting successful co-immunoprecipitation experiments with POI-myc-His fusion proteins.

Moreover, I figured the signal sequence will be cleaved by signal peptidases which will leave the c-myc tag exposed on N-terminus with only an extra residue. I don't think that one extra residue will create a problem.

Here's the tool I used to figure out where the signal sequence will be cleaved: http://www.cbs.dtu.dk/services/SignalP/

Here are the references for POI-myc-His experiments:

  • DOI: 10.1016/j.cub.2006.08.085
  • DOI: 10.1073/pnas.97.2.668

LUMIER: A Discovery Tool for Mammalian Protein Interaction Networks

Protein-protein interactions (PPIs) play an essential role in all biological processes. In vivo, PPIs occur dynamically and depend on extracellular cues. To discover novel protein-protein interactions in mammalian cells, we developed a high-throughput automated technology called LUMIER (LUminescence-based Mammalian IntERactome). In this approach, we co-express a Luciferase (LUC)-tagged fusion protein along with a Flag-tagged protein in an efficiently transfectable cell line such as HEK-293T cells. The interaction between the two proteins is determined by co-immunoprecipitation using an anti-Flag antibody, and the presence of the LUC-tagged interactor in the complex is subsequently detected via its luciferase activity. LUMIER can easily detect transmembrane protein partners, interactions that are signaling- or splice isoform-dependent, as well as those that may occur only in the presence of posttranslational modifications. Using various collections of Flag-tagged proteins, we have generated protein interaction networks for several TGF-β family receptors, Wnt pathway members, and have systematically analyzed the effect of neural-specific alternative splicing on protein interaction networks. The results have provided important insights into the physiological and functional relevance of some of the novel interactions found. LUMIER is highly scalable and can be used for both low- and high-throughput strategies. LUMIER is thus a valuable tool for the identification and characterization of dynamically regulated PPIs in mammalian systems. Here, we describe a manual version of LUMIER in a 96-well format that can be easily implemented in any laboratory.

Keywords: Binary complex LUMIER Mammalian cells Protein–protein interaction Signaling pathways Ternary complex Transmembrane proteins.


Fusion Proteins

Uses of Fusion Proteins

The technique of creating fusion proteins has been extended to other fusion partners, and additional uses have been developed for the fusion partner. Three of the most important uses of fusion proteins are: as aids in the purification of cloned genes, as reporters of expression level, and as histochemical tags to enable visualization of the location of proteins in a cell, tissue, or organism.

For purification, a protein that can be easily and conveniently purified by affinity chromatography is fused to a protein that the researcher wishes to study. A number of proteins and peptides have been used for this purpose, including staphylococcus protein A, glutathione-S-transferase, maltose-binding protein, cellulose-binding protein, chitin-binding domain, thioredoxin, strepavidin, RNaseI, polyhistidine, human growth hormone, ubiquitin, and antibody epitopes.

The proteins used most often as fusion partners for reporter constructs are β-galactosidase, luciferase, and green fluorescent protein (GFP). β-galactosidase has the advantage of numerous commercially available substrates, including some that produce a colored product and some that lead to the production of light. Luciferase and GFP both produce light, and can be visualized directly or quantitated using a luminometer or a fluorometer, respectively. GFP has an advantage in that it does not require a substrate, whereas luciferase requires its substrate, luciferin, as well as ATP, O2, and Mg 2+ . GFP emits green light when excited by blue or UV light, and in many cases can be used on live, intact cells and organisms.

A useful extension of fusion proteins as reporters is the two-hybrid system. In this method, two separate fusions are employed to test for interaction between two proteins, where binding of the two proteins brings together their fusion partners and results in activated transcription of a reporter gene.


Fusion of DARPin to Aldolase Enables Visualization of Small Protein by Cryo-EM

Solving protein structures by single-particle cryoelectron microscopy (cryo-EM) has become a crucial tool in structural biology. While exciting progress is being made toward the visualization of small macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of cryo-EM. To overcome this problem, we implemented a platform strategy in which a small protein target was rigidly attached to a large, symmetric base via a selectable adapter. Of our seven designs, the best construct used a designed ankyrin repeat protein (DARPin) rigidly fused to tetrameric rabbit muscle aldolase through a helical linker. The DARPin retained its ability to bind its target: GFP. We solved the structure of this complex to 3.0 Å resolution overall, with 5-8 Å resolution in the GFP region. As flexibility in the DARPin position limited the overall resolution of the target, we describe strategies to rigidify this element.

Keywords: CryoEM DARPin Electron cryo-microscopy Single-particle analysis aldolase artificial protein cryo-electron microscopy platform protein design.


Development of an Antigen-Antibody Co-Display System for Detecting Interaction of G-Protein-Coupled Receptors and Single-Chain Variable Fragments

G-protein-coupled receptors (GPCRs), especially chemokine receptors, are ideal targets for monoclonal antibody drugs. Considering the special multi-pass transmembrane structure of GPCR, it is often a laborious job to obtain antibody information about off-targets and epitopes on antigens. To accelerate the process, a rapid and simple method needs to be developed. The split-ubiquitin-based yeast two hybrid system (YTH) was used as a blue script for a new method. By fusing with transmembrane peptides, scFv antibodies were designed to be anchored on the cytomembrane, where the GPCR was co-displayed as well. The coupled split-ubiquitin system transformed the scFv-GPCR interaction signal into the expression of reporter genes. By optimizing the topological structure of scFv fusion protein and key elements, including signal peptides, transmembrane peptides, and flexible linkers, a system named Antigen-Antibody Co-Display (AACD) was established, which rapidly detected the interactions between antibodies and their target GPCRs, CXCR4 and CXCR5, while also determining the off-target antibodies and antibody-associated epitopes. The AACD system can rapidly determine the association between GPCRs and their candidate antibodies and shorten the research period for off-target detection and epitope identification. This system should improve the process of GPCR antibody development and provide a new strategy for GPCRs antibody screening.

Keywords: G protein coupled receptor antibodies epitope off-targets yeast two hybrid.

Conflict of interest statement

The funders had no role in the design of the study in the collection, analyses, or interpretation of data in the writing of the manuscript or in the decision to publish the results.


Protogenin defines a transition stage during embryonic neurogenesis and prevents precocious neuronal differentiation.
Fann MJ
The Journal of neuroscience : the official journal of the Society for Neuroscience 30.12 (2010 Mar 24): 4428-39.

Regulation of Fasciclin II and synaptic terminal development by the splicing factor beag.
McCabe BD
The Journal of neuroscience : the official journal of the Society for Neuroscience 32.20 (2012 May 16): 7058-73.

Sc65 is a novel endoplasmic reticulum protein that regulates bone mass homeostasis.
Morello R
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 29.3 (2014 Mar): 666-75.

Neuronal nitric oxide synthase-dependent S-nitrosylation of gephyrin regulates gephyrin clustering at GABAergic synapses.
Schwarz G
The Journal of neuroscience : the official journal of the Society for Neuroscience 34.23 (2014 Jun 4): 7763-8.

Protogenin defines a transition stage during embryonic neurogenesis and prevents precocious neuronal differentiation.
Fann MJ
The Journal of neuroscience : the official journal of the Society for Neuroscience 30.12 (2010 Mar 24): 4428-39.

Syne proteins anchor muscle nuclei at the neuromuscular junction.
Han M
Proceedings of the National Academy of Sciences of the United States of America 102.12 (2005 Mar 22): 4359-64.

Regulation of Fasciclin II and synaptic terminal development by the splicing factor beag.
McCabe BD
The Journal of neuroscience : the official journal of the Society for Neuroscience 32.20 (2012 May 16): 7058-73.


Applications

  • Purify or immunoprecipitate myc-tagged fusion proteins from cell lysates
  • Identify myc-tagged proteins
  • Perform high-throughput protein-protein interaction studies

Additional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.

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MATERIALS AND METHODS

Cell culture and transfections

All cell lines were incubated in humidified atmosphere at 37°C with 5% CO2. P493-6 and Jurkat T cells were cultivated in RPMI medium and HEK293, HEK293T, U2OS and HeLa cells were grown in DMEM+Glutamax medium, both supplemented with 10% heat-inactivated fetal calf serum and 1% penicillin/streptomycin (P/S). For growth-arrest and MYC suppression, P493-6 B cells were seeded at 5 × 10 5 /ml and treated with 0.1 μg/ml tetracycline for 72 h. To induce MYC expression, tetracycline was removed by washing the cells twice with phosphate buffered saline (PBS) and adding fresh RPMI medium containing 10% FCS (+MYC). Control cells were maintained in tetracycline-containing medium until harvest (-MYC).

Transfections of plasmids were carried out using the calcium phosphate method ( 35, 36). Experiments were harvested 36 to 48 h after transfection when only protein-expressing plasmids were used. Experiments with plasmids expressing short-hairpin RNAs (pSuper) were harvested 72 h after transfection. Transient transfections with Dharmacon siRNAs were carried out using HiPerfect (Qiagen, Hilden Germany) transfection reagent according to the manufacturer's instructions at a final concentration of 15–20 nM.

Dharmacon siRNA pools (Thermo Scientific)

Control: siGENOME Non-Targeting siRNA Pool #2 (D-001206-14)

ASH2L: SMARTpool siGENOME ASH2L siRNA (M-019831-01)

MYC: SMARTpool siGENOME MYC siRNA (M-003282-07)

Dharmacon Set of 4 single siRNAs (Thermo Scientific)

ASH2L: siGENOME ASH2L siRNA (MQ-019831-01)

MYC: siGENOME MYC siRNA (MQ-003282-07)

The four individual siOligos were tested for knockdown efficiency (data not shown). The two most efficient siOligos were then used in additional control experiments (shown in Supplementary Figures S6 and S7).

Plasmids, siRNAs and recombinant proteins

pcDNA3-Flag-MYC-1-439 (wt), -1-410, -1-350, -1-180, -101-439, -148-439, -178-439, -Δ103-263, -Δ265-329, -Δ265-367, -Δ319-341 and pCGN-HA-MYC-1-439 (wt), -1-366, -1-293, -1-220, -221-439, -294-439, -367-439 were kindly provided by W. Tansey ( 37). Plasmids expressing ASH2L were described previously ( 38). Mutants were generated using standard cloning techniques. The following pSuper ( 39) constructs were used: pSuper-ASH2L 5’-GGATCTCACTTACCGCCCT pSuper-Control (ASH2Lmut) 5’-CCCTGCAGATCCATGCTT. pcDNA3-Flag-MLL2 653 (Addgene 11017), pcDNA3-Flag-WDR5 (Addgene 15552) and pcDNA3 RbBP5 (Addgene 15550) plasmids were used for the expression of the KMT2 complex components.

Recombinant proteins were produced in Escherichia coli (E. coli) BL21(DE)pLysS as GST-fusion or His6-fusion proteins using the following constructs pGEX4T3-ASH2L-wt, -1-121, -1-279, -1-394, -1-444, pGEX2T-MYC-1-262 (N262), -1-156, -263-439, pGEX3X-MAX, pGEX2T-YY1, pGEX4T3-H3-N, pQE30-ASH2-1-387, pRSET-His6-WDR5 ( 40). Transformed bacteria were grown to an OD of 0.7–0.9 and protein expression was induced with 0.4 mM IPTG either at 37°C for 4 h or at 21°C for 16 h. Bacterial pellets were lysed in buffer A (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5% (v/v) Nonidet P40, 5 mM EDTA, 10 mM DTT, 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 1% (v/v) Aprotinin), sonicated, and centrifuged at 10.000 x g at 4°C for 30 min. GST-fusion proteins were separated from the cell lysate with glutathione-agarose (Sigma-Aldrich) and eluted from the beads with 10 mM glutathione in buffer A.

Maltose-binding protein-MYC fusion (MBP-MYC) and MBP ( 41) were purified from E. coli BL21(DE)pLysS grown in NZC-medium (1% NZ-Amine A, 0.5% (w/v) NaCl, 0.2% (w/v) MgCl2, 0.2% glucose) induced at OD 0.5 with 0.3 mM IPTG at 30°C for 4 h. Cells were lysed in buffer C (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.2 mM PMSF, 1% (v/v) Aprotinin), sonicated, and centrifuged at 10,000 x g at 4°C for 30 min. MBP proteins were separated from the cell lysate with amylose-agarose (New England Biolabs) and eluted from the beads with 10 mM maltose in buffer A. Core histones and recombinant histone H3 (New England Biolabs) and GST-Histone H3 N’-terminal-tails (provided by Y. Shinkai) ( 40) were used as substrates.

GST-pull-down assays

For binding reactions, 3 μg GST or GST-fusion proteins were bound to glutathione–agarose beads and incubated with [ 35 S]methionine-labeled, in vitro transcribed and translated proteins in binding buffer or with 3 μg of recombinant His6-fusion proteins ( 42). For in vitro translation/transcription, the TNT Quick Coupled Transcription/Translation System (Promega) was used with the plasmids pcDNA3-Flag-MYC and pcDNA3-Flag-ASH2L.

Co-immunoprecipitation

For in cell interaction assays, whole-cell lysates from 2 × 10 7 cells were prepared in F-buffer (10 mM Tris-HCl, pH 7.05, 50 mM NaCl, 30 mM Na3PO4, 50 mM NaF, 5 μM ZnCl, 100 μM Na3VO4, 1% Triton X-100, 1 mM PMSF, 5 units/ml α2-macroglobulin, 2.5 units/ml pepstatin A, 2.5 units/ml leupeptin, 0.15 mM benzamidin). Co-immunoprecipitated proteins were detected by western blot analysis ( 43, 44).

Histone methyltransferase assay

Whole-cell lysates from 3 × 10 7 cells were prepared in F-buffer to immunoprecipitate MYC or ASH2L and the associated MTase. MYC- and ASH2L-associated MTase was measured with 5 μg core histones or GST-H3 N-terminal tails as substrates in the presence of 1.5 μl [ 3 H]-SAM (Perkin-Elmer Life Sciences, NET-155H, 0.55 mCi/ml, 79.8 Ci/mmol) at 30°C for 30 min. Modified proteins were visualized by SDS-PAGE and autoradiogaphy. Site specificity and MYC-associated methyltransferase activity was measured with 1 μg recombinant histone H3 in the presence of 100 μM SAM at 30°C for 1 h. Histone H3 methylation was detected by immunoblotting with different H3K4 and H3K9 methyl-specific antibodies.

Immunofluorescence staining

HeLa cells were seeded onto coverslips, transiently transfected with pSuper-ASH2L and fixed in PBS containing 3.7% paraformaldehyde 72 h after transfection. Cells were permeabilized in PBS with 0.2% Triton X-100 at room temperature for 5 min and stained with the ASH2L-specific rat mAb 4C5 and the H3K4me3-specific rabbit polyclonal antibody ab8898 (abcam) at 37°C for 45 min. Anti-rat-Cy3 and anti-rabbit-Cy2 secondary antibodies were used at 37°C for 30 min. Coverslips were mounted with Moviol (Merck) in PBS containing 2.5% N-propylgallate (Sigma). For the proximity ligation assay (PLA), the DuolinkII fluorescence system was used in conjunction with the Probemaker Plus kit for labeling of the secondary anti-rat antibody according to the manufacturer's instructions (Olink Bioscience) ( 45).

Antibodies

The following antibodies were used for IP, chromatin immunoprecipitations (ChIP), and western blot: rabbit anti-MYC (N262, sc-764 C19, sc-788, both Santa Cruz IG13, 022Y, 1236 all three provided by L.-G. Larsson), rabbit anti-MAX (C17, sc-197, Santa Cruz), rat anti-MXD1 (5F4), mouse anti-nucleolin (MS-3, sc-8031, Santa Cruz) rabbit anti-ASH2L (A300–489A, Bethyl sera 548 and 549 (antigen ASH2L-1-444)), rat anti-ASH2L (4C5 and 4B5 (antigen ASH2L-1-279)), mouse anti-WDR5 (ab56919, abcam), rabbit anti-RbBP5 (A300-109A, Bethyl), rabbit anti-H3 (ab1791, abcam), rabbit anti-H3ac (06-599, Millipore), rabbit anti-H3K9ac (ab4441, abcam), rabbit anti-H3K27ac (ab4729, abcam), rabbit anti-H3K4me3 (ab8580, abcam), rabbit anti-H3K4me2 (07-030, Millipore), rabbit anti-H3K4me1 (ab8895, abcam), rabbit anti-H3K9me3 (ab8898, abcam), rabbit H3K9me2 (07-212, Millipore), rabbit anti-H3K9me1 (ab9045, abcam), rabbit anti-H3K27me3 (ab6002, abcam), rabbit anti-CBP (C-20, sc-583, Santa Cruz), rabbit anti-p300 (N-15, sc-584, Santa Cruz), rabbit IgG (Kch-504-250, Diagenode), mouse anti-actin (MP Biomedicals), rat anti-HA (3F10, Roche), mouse anti-Flag (M2, Sigma-Aldrich), rabbit anti-caspase 5 (2157, was kindly provided by A. Krippner-Heidenreich).

ChIP- and Re-ChIP-qPCR

ChIP-qPCR assays were performed with the Diagenode OneDay ChIP kit according to the manufacturer's instructions. P493-6 and HEK293T cells were cross-linked with 1% formaldehyde (by adding 37% formaldehyde to the medium) on a shaking platform for 10 min at room temperature and quenched for 5 min by adding 125 mM glycine. Cells were lysed in 100 μl lysis buffer (2 × 10 7 P493-6 cells and 1×10 7 HEK293T cells) and chromatin was sheared for 15 min using a Bioruptor (Diagenode). Immunoprecipitation was performed with 100 μg chromatin (input) and 2 μg antibody. DNA was purified and the quantitative polymerase chain reaction (qPCR) was performed with the following primer pairs:

Ctrl pp4 (for): 5’-GCGTGACTCAAATTGTGTGTGCCT-3’

Ctrl pp4 (rev): 5’-ATCAAGCATTCAGCAGCGTTC CA-3’

CCND2 prom1 (for): 5’-CCCCTTCCTCCTGGAGTGAAATAC-3’

CCND2 prom2 (rev): 5’-CGTGCTCTAACGCATCCTTGAGTC-3’

CCND2 Ex1 (for): 5’-GGGAGAGCGAGACCAGTTTTAAG-3’

CCND2 Ex1 (rev): 5’-CCTTTGGCTAAATAGGGGGTTTTC-3’

ODC E-box (for): 5’-ATCACTTCCAGGTCCCTTGCAC-3’

ODC E-box (rev): 5’-TTCCACCTGGCGTTCAGTACCT-3’

NCL In1 (for): 5’-TGGGCCGGGAAATGGCGGTA-3’

NCL In1 (rev): 5’-TAGGCCACCACGTGCCCGAA-3’

For Re-ChIP experiments the first IP was performed with 100–200 μg chromatin (input). Chromatin was released from beads using release buffer (TE buffer, pH 7.5, 1% SDS, 10 mM DTT) for 30 min at 37°C and diluted in 4 volumes ChIP buffer (Diagenode OneDay ChIP kit, with protease inhibitor cocktail and 10 mM iodoacetamide). Then, 2 μg of antibody were added for the second immunoprecipitation, followed by DNA purification and qPCR. PCR was performed as described below.

The DNA was purified from one tenth of the input chromatin that was used for the ChIPs, diluted two-fold and used for qPCR amplification. The dilution factor was included in the % input calculation of all the qPCR results.

QRT-PCR

Total RNA was extracted using RNeasy Mini Kit (Qiagen) and DNase digestion (Qiagen) according to the manufacturer's instructions. 1 μg of RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) and analyzed by qPCR with SensiMix SYBR Green Mix (Bioline) in a Rotor-Gene Q (Qiagen) machine.

The following primer pairs were used for amplification:

β-glucuronidase (GUS) for: 5’-CTCATTTGGAATTTTGCCGATT-3’,

β-glucuronidase (GUS) rev: 5’-CCGAGTGAAGATCCCCTTTTTA-3’

QuantiTect primer assays (Qiagen): MYC (Hs_Myc_1_SG), ASH2L (Hs_ASH2L_1_SG), CCND2 (Hs_CCND2_1_SG), ODC (Hs_ODC1_1_SG), NCL (Hs_NCL_1_SG). Relative mRNA expression was calculated by the comparative ΔΔCt method and normalized to the housekeeping gene GUS using Rotor-Gene Q software.

P493-6 cells were harvested by centrifugation, washed with PBS and fixed by adding 100% methanol at -20°C. After RNase A treatment (20 μg/ml) cells were stained with propidium iodide (PI, 50 μg/ml) for 15 min at room temperature and analyzed using a FACSCanto II (BD Biosciences).

Bioinformatics analysis of publicly available ChIP-Seq data

Public data from ChIP experiments followed by massively parallel DNA sequencing (ChIP-Seq) was used to analyze the occupancy of the factors MYC, WDR5 and POL II and the occurrence of the histone modifications H3K4me3, H3K4me1 and H3K27me3 in normal human epidermal keratinocytes (NHEK). Data from histone modifications, POL II, MYC and controls were obtained from the ENCODE project (GEO accession GSE29611, GSM748557). WDR5 ChIP-Seq was obtained from published findings ( 46) (GEO accession GSE42180). No read alignments were provided in GEO for WDR5 and MYC. For those, the alignments from original sequence reads were performed using Burrows-Wheeler Alignment tool with default parameters ( 47). All alignments were based on the human reference genome Hg19. Peak calling was performed with MACS ( 48) with a p-value of 10 −5 and the ChIP-Seq control as input.

Definition of regulatory features

Publicly available data for histone modifications were combined to define regulatory regions in NHEK cells: active promoters (both H3K27ac and H3K4me3 peaks overlapping with the core promoter, 200 bps upstream of a gene transcription start site (TSS)), active enhancers (regions distant from core promoters with peaks of both H3K4me1 and H3K27ac), poised promoters (H3K4me3 and H3K27me3 ( 49)), poised enhancers (H3K4me1, lack of H3K27ac ( 50)) and closed chromatin regions (only H3K27me3). Regions not fitting any of these criteria were indicated as ‘other’. bedTools ( 51) was used to find the regions of overlap between these sites that contain histone with specific modifications and the TSS of genes. Gene TSS positions were based on Hg19 RefSeq annotation obtained from USCD Table Browser. Additionally, the overlap of these regions with POL II ChIP-Seq peaks was evaluated. As a last step, the peaks from MYC, WDR5 and regions containing both MYC and WDR5 peaks were related to the above-mentioned regulatory features (see Supplementary Tables S1 and S2 for statistics).

Analysis of transcription factor binding sites

To analyze the presence of binding sites for MYC and ASH2L inside ChIP-Seq peaks, the summit regions of the Chip-Seq peaks (positions with highest reads within a peak as provided by MACS), extended by 125 bps in each direction, were inspected. This procedure ensures that all peaks possess the same size and avoids biasing the binding site statistics. The choice of peaks with a size of 250 bps is in accordance with previously published work ( 52). Note that other choices of peak sizes (500, 100 and 80 bps) resulted in similar qualitative results (data not shown). As background, random genomic regions with the same characteristics of the ChIP-Seq peaks were obtained. The MYC position weight matrix (MA0147.1) from the Jaspar database ( 53) and the ASH2L position weight matrix from ( 54) were used. Biopython ( 55) was employed to perform binding site searches. Functions provided by the Motif class were applied to calculate a bit-score based on the application of a position Weight Matrix in the ChIP-Seq peaks. An empirical statistical test based on dynamic programming was used to define a bit-score threshold with a false discovery rate of 0.0001 ( 56). The number of MYC, WDR5 and both MYC and WDR5 ChIP-Seq peaks with at least one binding site motif were counted and a Fisher's Exact test performed to evaluate whether the proportion of peaks with binding sites is higher than in the background regions (Supplementary Table S3). Using the same analysis, but classifying the MYC or WDR5 peaks that belong to the previously described regulatory features, no relation between the proportion of binding sites and the regulatory features surrounding the peaks was detected (not shown). The analysis was performed with the Regulatory Genomics Toolbox available at https://code.google.com/p/reg-gen/.

Analysis of gene expression

To evaluate if regions of ChIP-Seq peaks and NHEK expression data from RNA-Seq experiments are related, the alignment of RNA-Seq reads from the ENCODE project (GSM958736) was analyzed. The summits of peaks of MYC, WDR5 and both MYC and WDR5 were extended by 1000 bps to capture the expression of neighboring genes. The number of aligned reads inside the ChIP-Seq peaks was counted and normalized by the size of the peaks. In addition, the same analysis was performed considering only ChIP-Seq peaks overlapping with the region 200 bps upstream of a TSS.


C-myc/anti-myc Ab Interaction with Fusion Proteins - Biology

Integrin ligand binding induces a signaling complex formation via the direct association of the docking protein p130 Cas (Cas) with diverse molecules. We report here that the 14-3-3ζ protein interacts with Cas in the yeast two-hybrid assay. We also found that the two proteins associate in mammalian cells and that this interaction takes place in a phosphoserine-dependent manner, because treatment of Cas with a serine phosphatase greatly reduced its ability to bind 14-3-3ζ. Furthermore, the Cas-14-3-3ζ interaction was found to be regulated by integrin-mediated cell adhesion. Thus, when cells are detached from the extracellular matrix, the binding of Cas to 14-3-3ζ is greatly diminished, whereas replating the cells onto fibronectin rapidly induces the association. Consistent with these results, we found that the subcellular localization of Cas and 14-3-3 is also regulated by integrin ligand binding and that the two proteins display a significant co-localization during cell attachment to the extracellular matrix. In conclusion, our results demonstrate that 14-3-3 proteins participate in integrin-activated signaling pathways through their interaction with Cas, which, in turn, may contribute to important biological responses regulated by cell adhesion to the extracellular matrix.


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