Multisite-Directed Mutagenesis.

This protocol, suitable for both general and close-proximity mutagenesis, includes a simple and rapid procedure that combines polymerase chain reaction (PCR), DpnI digestion, and overlap extension. The key point of this approach is the use of overlap extension to form a circular DNA plasmid with mutations without the need for phosphorylated primers or ligase reactions. Essentially, during the first round of PCR, the new DNA is synthesized with nicks between the 3' ends of the synthesized DNA and the 5' ends of the first pair of primers. During successive rounds of PCR, a new pair of mutagenic oligonucleotides leads to the synthesis of two DNA segments that anneal together with the overlap sequence inside the two primers. This new mutated molecule also contains nicks but at different positions compared with those formed in the first round of PCR that had been "repaired" by overlap extension. Mutations can be introduced successfully by this method. Finally, the circular DNA is transformed into E. coli cells, where the nicks are ligated into a circular plasmid. One important requirement is that the parental plasmid carrying the target gene needs to be methylated by Dam methyltransferase or purified from Dam+ E. coli (i.e., DH5a).

Methods for In Vitro Mutagenesis.

Several different methodologies for mutagenesis have been developed to introduce mutations at predetermined sites or regions within mammalian genes. These methods of in vitro mutagenesis have had a transforming effect on the understanding of functions of protein, transcription regulatory elements, and noncoding RNAs and are now integral to molecular biology investigations. In this introduction, we summarize the most commonly used experimental approaches for mutagenesis and their major applications.

Random Scanning Mutagenesis.

In vitro oligonucleotide and polymerase chain reaction (PCR)-based mutagenesis is generally used for altering the nucleotide sequence of genes to study their functional importance and the products they encode. A thorough approach to this problem is to systematically change each successive amino acid residue in the protein to alanine (i.e., alanine-scanning mutagenesis) or to a limited number of alternative amino acids. Although these strategies can provide useful information, it is sometimes desirable to test a broader spectrum of amino acid changes at the targeted positions. "Random scanning mutagenesis" was developed to examine the functional importance of individual amino acid residues in the conserved structural motif of human immunodeficiency virus (HIV) reverse transcriptase, and this protocol is adapted from that method. This strategy is an oligonucleotide-based method for generating all 19 possible replacements at individual amino acid sites within a protein.

Megaprimer Polymerase Chain Reaction (PCR)-Based Mutagenesis.

The megaprimer method is a simple and versatile approach that can be adopted to create a single mutation in a specific target region as well as to create site-specific insertions, deletions, and gene fusions. This method uses three oligonucleotide primers, two rounds of polymerase chain reaction (PCR), and a DNA template containing the gene to be mutated. The first round of PCR generates a fragment with the desired mutation introduced by using one of the flanking primers and the mutant primer. This amplified fragment-the megaprimer-is used in the second PCR along with the remaining external primer to amplify a longer region of the template DNA. The final product is purified and can be cloned into an appropriate vector. By designing flanking primers with universal restriction site sequences, compatible with the vector of choice, it is possible to create different mutant clones by changing only the mutant primer. Recently, this approach has been improved by the use of forward and reverse flanking primers with significantly different melting temperatures. This allows researchers to perform both PCR steps in a single tube. This protocol has been successfully applied on templates with either low or high G + C content to amplify megaprimers 71-800 bp in length and final products ranging from 400 to 2500 bp.

Oligonucleotide-Directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis).

In this method, two oligonucleotide primers are hybridized to the same strand of a denatured double-stranded recombinant plasmid. One primer (the mutagenic primer) introduces the desired mutation into the target sequences, whereas the second primer carries a mutation that destroys a unique restriction site (called "unique site elimination," or USE) in the plasmid. The product of the first part of the method is a heteroduplex plasmid consisting of a wild-type parental strand and a new full-length strand that carries the desired mutation but no longer contains the unique restriction site. In the second phase of the method, the mixed population is incubated with the restriction enzyme that cleaves the unique site. The wild-type molecules are linearized, and the mutated plasmids are resistant to digestion. The mixture of circular heteroduplex DNA and linear wild-type DNA is then used to transform a strain of Escherichia coli that is deficient in repair of mismatched bases. The circular heteroduplex molecules begin to replicate while many of the linear wild-type molecules are unable to reestablish themselves in E. coli cells. Because the mismatched bases are not repaired, the first round of replication generates a wild-type plasmid that carries the original restriction site and a mutated plasmid that does not. DNA from the first set of transformants is recovered, digested once more with the same restriction enzyme to linearize the wild-type molecules, and then used to transform a standard laboratory strain of E. coli.

Saturation Mutagenesis by Codon Cassette Insertion.

Saturation mutagenesis by cassette insertion introduces a library of site-specific changes into a specific DNA sequence within a target gene and is especially useful for analyzing the effect of specific residues on the structure and function of a protein. In this protocol, a set of 11 universal oligodeoxyribonucleotide cassettes is used to generate mutations. The major advantage of this method is that a single set of mutagenic codon cassettes can be used to insert codons encoding all possible amino acids at any predetermined site within a gene. Each of the 11 cassettes contains two recognition sequences for SapI, a restriction enzyme that cleaves outside of its recognition sequence. The recognition sequences for SapI are arranged in opposite orientations and are separated by a central spacer. At the end of each cassette is a 3-bp direct repeat, positioned such that the sites of SapI cleavage bracket each repeat. Cleavage by SapI will result in the generation of three base-cohesive single-stranded ends on the end of the cassette. These three base-cohesive single-stranded ends can then be ligated together to regenerate the original 3-bp direct repeat, while excising the central spacer. It is this 3-bp repeat sequence that is ultimately incorporated into the template. By substituting the 3-bp direct repeats in the universal cassette with other sequences, one can essentially generate all possible amino acid substitutions.

Creating Insertions or Deletions Using Overlap Extension Polymerase Chain Reaction (PCR) Mutagenesis.

Overlap extension polymerase chain reaction (PCR) mutagenesis can be used for the generation of a specific point mutation, insertion, or deletion within a particular DNA sequence of interest. It requires relatively little preparation compared with other mutagenesis methods and does not require the use of restriction enzymes. Because of its versatility, the method has become widely used. Unlike methods of random mutagenesis, directed mutagenesis requires that the researcher already have a specific mutation in mind to implement. Traditional overlap extension PCR mutagenesis protocols remain limited in several critical ways, especially when it comes to generating insertions and deletions. For example, traditional protocols require that all sequence alterations be embedded within the primer itself, which makes it difficult to make insertions >30 nt. This protocol describes an overlap extension PCR mutagenesis method that is more versatile than its predecessors. Using this method, one can essentially make insertions and deletions of any size at any position within a given DNA sequence. To generate an insertion mutation, first prepare an insertion fragment and two flanking fragments by PCR. In the secondary PCR, the insertion fragment is recombined with two flanking fragments derived from the original template. This method can also be used to generate deletions, which is discussed in the latter part of the protocol.

Altered ß-Lactamase Selection Approach for Site-Directed Mutagenesis.

Many protocols exist to perform site-directed mutagenesis, and here we present one of the more commonly used ones-site-directed mutagenesis by altered ß-lactamase selection. ß-Lactamase is an enzyme that cleaves ampicillin, rendering it impotent to bacteria. Certain mutations in the active site of ß-lactamase can alter the substrate specificity of the enzyme and allow it to have increased hydrolytic activity for the cephalosporin family of antibiotics, a property not shared by wild-type lactamases. E. coli cells carrying the ß-lactamase triple mutant G238S:E240:R241G show increased resistance to cefotaxime and ceftriaxone, two cephalosporins, compared with wild-type cells. This protocol takes advantage of this property to select for plasmids that have undergone site-directed mutagenesis.

Random Mutagenesis Using Error-Prone DNA Polymerases.

"Random mutagenesis" is a technique that allows researchers to develop large libraries of variants of a particular DNA sequence. Once developed, these libraries can then be used for several purposes, including structure-function and directed evolution studies. Random mutagenesis is different from other mutagenesis techniques in that it does not require the researcher to have any prior knowledge about the structural properties of the DNA sequence being targeted, thus allowing for the unbiased discovery of novel or beneficial mutations. For this reason, random mutagenesis is especially useful for protein evolution studies. This protocol describes mutagenic replication in vitro by a low-fidelity DNA polymerase followed by selective polymerase chain reaction (PCR) amplification of the newly mutated sequences. The initial mutagenic DNA replication step is accomplished by heat-denaturing the template DNA and annealing primers possessing 5' extensions that are not complementary to the template. The purpose of the noncomplementary extensions on the primers is to allow for future selection of only the mutant strands. DNA replication is then performed by a low-fidelity DNA polymerase of choice (polymerase ß, η, or ι, or any combination of the three). After mutations have been incorporated into the template, the mutagenized strands are then selectively amplified using PCR. Selective amplification of the mutant strands is accomplished by performing a PCR procedure consisting of a first cycle with a low hybridization temperature followed by subsequent selection cycles under higher hybridization temperatures that do not allow amplification of the original unmutagenized template.

In Vitro Mutagenesis Using Double-Stranded DNA Templates: Selection of Mutants with DpnI.

In this protocol, two oligonucleotides are used to prime DNA synthesis by a high-fidelity polymerase on a denatured plasmid template. The two oligonucleotides both contain the desired mutation and have the same starting and ending positions on opposite strands of the plasmid DNA. The entire lengths of both strands of the plasmid DNA are amplified in a linear fashion during several rounds of thermal cycling, generating a mutated plasmid containing staggered nicks on opposite strands. Because of the amount of template DNA used in the amplification reaction, the background of transformed colonies containing wild-type plasmid DNA can be quite high unless steps are taken to enrich for mutant molecules. In this protocol, the products of the linear amplification reaction are treated with the restriction enzyme DpnI, which specifically cleaves fully methylated GMe6ATC sequences. DpnI will therefore digest the bacterially generated DNA used as template for amplification, but it will not digest DNA synthesized during the course of the reaction in vitro. DpnI-resistant molecules, which are rich in the desired mutants, are recovered by transforming E. coli cells to antibiotic resistance. Because the method works well with virtually any plasmid of moderate size (<7 kb), it can be used to introduce mutations directly into full-length cDNAs and eliminates the need for subcloning into specialized vectors.

Large-Scale RNA Interference Screening to Identify Transcriptional Regulators of a Tumor Suppressor Gene.

RNA interference (RNAi) is a powerful research tool that can be used to silence the expression of a specific gene. In the past several years, RNAi has provided the opportunity to identify factors and pathways involved in complex biological processes by performing unbiased loss-of-function screens on a genome-wide scale. Here we describe a genome-wide RNAi screening strategy to identify factors that regulates epigenetic silencing of a specific tumor suppressor gene, using RASSF1A as an example. The approach we describe is a general RNAi screening strategy that can be applied to identify other factors that drive and/or maintain epigenetic modifications on specific genes, including cancer-related genes.

Interferon alpha-inducible protein 6 regulates NRASQ61K-induced melanomagenesis and growth.

Mutations in the NRAS oncogene are present in up to 20% of melanoma. Here, we show that interferon alpha-inducible protein 6 (IFI6) is necessary for NRASQ61K-induced transformation and melanoma growth. IFI6 was transcriptionally upregulated by NRASQ61K, and knockdown of IFI6 resulted in DNA replication stress due to dysregulated DNA replication via E2F2. This stress consequentially inhibited cellular transformation and melanoma growth via senescence or apoptosis induction depending on the RB and p53 pathway status of the cells. NRAS-mutant melanoma were significantly more resistant to the cytotoxic effects of DNA replication stress-inducing drugs, and knockdown of IFI6 increased sensitivity to these drugs. Pharmacological inhibition of IFI6 expression by the MEK inhibitor trametinib, when combined with DNA replication stress-inducing drugs, blocked NRAS-mutant melanoma growth. Collectively, we demonstrate that IFI6, via E2F2 regulates DNA replication and melanoma development and growth, and this pathway can be pharmacologically targeted to inhibit NRAS-mutant melanoma.

Oncogenic EGFR Represses the TET1 DNA Demethylase to Induce Silencing of Tumor Suppressors in Cancer Cells.

Oncogene-induced DNA methylation-mediated transcriptional silencing of tumor suppressors frequently occurs in cancer, but the mechanism and functional role of this silencing in oncogenesis are not fully understood. Here, we show that oncogenic epidermal growth factor receptor (EGFR) induces silencing of multiple unrelated tumor suppressors in lung adenocarcinomas and glioblastomas by inhibiting the DNA demethylase TET oncogene family member 1 (TET1) via the C/EBPα transcription factor. After oncogenic EGFR inhibition, TET1 binds to tumor suppressor promoters and induces their re-expression through active DNA demethylation. Ectopic expression of TET1 potently inhibits lung and glioblastoma tumor growth, and TET1 knockdown confers resistance to EGFR inhibitors in lung cancer cells. Lung cancer samples exhibited reduced TET1 expression or TET1 cytoplasmic localization in the majority of cases. Collectively, these results identify a conserved pathway of oncogenic EGFR-induced DNA methylation-mediated transcriptional silencing of tumor suppressors that may have therapeutic benefits for oncogenic EGFR-mediated lung cancers and glioblastomas.

miR-146a promotes the initiation and progression of melanoma by activating Notch signaling.

Oncogenic mutations in BRAF and NRAS occur in 70% of melanomas. In this study, we identify a microRNA, miR-146a, that is highly upregulated by oncogenic BRAF and NRAS. Expression of miR-146a increases the ability of human melanoma cells to proliferate in culture and form tumors in mice, whereas knockdown of miR-146a has the opposite effects. We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling. Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164). We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C. Analysis of melanoma cell lines and matched patient samples indicates that during melanoma progression pre-miR-146a/G is enriched relative to pre-miR-146a/C, resulting from a C-to-G somatic mutation in pre-miR-146a/C. Collectively, our results reveal a central role for miR-146a in the initiation and progression of melanoma. DOI: http://dx.doi.org/10.7554/eLife.01460.001.

IRF1 and NF-kB restore MHC class I-restricted tumor antigen processing and presentation to cytotoxic T cells in aggressive neuroblastoma.

Neuroblastoma (NB), the most common solid extracranial cancer of childhood, displays a remarkable low expression of Major Histocompatibility Complex class I (MHC-I) and Antigen Processing Machinery (APM) molecules, including Endoplasmic Reticulum (ER) Aminopeptidases, and poorly presents tumor antigens to Cytotoxic T Lymphocytes (CTL). We have previously shown that this is due to low expression of the transcription factor NF-kB p65. Herein, we show that not only NF-kB p65, but also the Interferon Regulatory Factor 1 (IRF1) and certain APM components are low in a subset of NB cell lines with aggressive features. Whereas single transfection with either IRF1, or NF-kB p65 is ineffective, co-transfection results in strong synergy and substantial reversion of the MHC-I/APM-low phenotype in all NB cell lines tested. Accordingly, linked immunohistochemistry expression patterns between nuclear IRF1 and p65 on the one hand, and MHC-I on the other hand, were observed in vivo. Absence and presence of the three molecules neatly segregated between high-grade and low-grade NB, respectively. Finally, APM reconstitution by double IRF1/p65 transfection rendered a NB cell line susceptible to killing by anti MAGE-A3 CTLs, lytic efficiency comparable to those seen upon IFN-γ treatment. This is the first demonstration that a complex immune escape phenotype can be rescued by reconstitution of a limited number of master regulatory genes. These findings provide molecular insight into defective MHC-I expression in NB cells and provide the rational for T cell-based immunotherapy in NB variants refractory to conventional therapy.

Genome-wide approaches for cancer gene discovery.

One of the central aims of cancer research is to identify and characterize cancer-causing alterations in cancer genomes. In recent years, unprecedented advances in genome-wide sequencing, functional genomics technologies for RNA interference screens and methods for evaluating three-dimensional chromatin organization in vivo have resulted in important discoveries regarding human cancer. The cancer-causing genes identified from these new genome-wide technologies have also provided opportunities for effective and personalized cancer therapy. In this review, we describe some of the most recent technologies for cancer gene discovery. We also provide specific examples in which these technologies have proven remarkably successful in uncovering important cancer-causing alterations.

Natural killer cells efficiently reject lymphoma silenced for the endoplasmic reticulum aminopeptidase associated with antigen processing.

The endoplasmic reticulum aminopeptidase ERAAP is involved in the final trimming of peptides for presentation by MHC class I (MHC-I) molecules. Herein, we show that ERAAP silencing results in MHC-I peptide-loading defects eliciting rejection of the murine T-cell lymphoma RMA in syngeneic mice. Although CD4 and CD8 T cells are also involved, rejection is mainly due to an immediate natural killer (NK) cell response and depends on the MHC-I-peptide repertoire because replacement of endogenous peptides with correctly trimmed, high-affinity peptides is sufficient to restore an NK-protective effect of MHC-I molecules through the Ly49C/I NK inhibitory receptors. At the crossroad between innate and adaptive immunity, ERAAP is therefore unique in its two-tiered ability to control tumor immunogenicity. Because a large fraction of human tumors express high levels of the homologous ERAP1 and/or ERAP2, the present findings highlight a convenient, novel target for cancer immunotherapy.

NF-kappaB, and not MYCN, regulates MHC class I and endoplasmic reticulum aminopeptidases in human neuroblastoma cells.

Neuroblastoma (NB) is the most common solid extracranial cancer of childhood. Amplification and overexpression of the MYCN oncogene characterize the most aggressive forms and are believed to severely downregulate MHC class I molecules by transcriptional inhibition of the p50 NF-kappaB subunit. In this study, we found that in human NB cell lines, high MYCN expression is not responsible for low MHC class I expression because neither transfection-mediated overexpression nor small interfering RNA suppression of MYCN affects MHC class I and p50 levels. Furthermore, we identified NF-kappaB as the immediate upstream regulator of MHC class I because the p65 NF-kappaB subunit binds MHC class I promoter in chromatin immunoprecipitation experiments, and MHC class I expression is enhanced by p65 transfection and reduced by (a) the chemical NF-kappaB inhibitor sulfasalazine, (b) a dominant-negative IKBalpha gene, and (c) p65 silencing. Moreover, we showed that the endoplasmic reticulum aminopeptidases ERAP1 and ERAP2, which generate MHC class I binding peptides, are regulated by NF-kappaB, contain functional NF-kappaB-binding elements in their promoters, and mimic MHC class I molecules in the expression pattern. Consistent with these findings, nuclear p65 was detected in NB cells that express MHC class I molecules in human NB specimens. Thus, the coordinated downregulation of MHC class I, ERAP1, and ERAP2 in aggressive NB cells is attributable to a low transcriptional availability of NF-kappaB, possibly due to an unknown suppressor other than MYCN.

Antagomir-17-5p abolishes the growth of therapy-resistant neuroblastoma through p21 and BIM.

We identified a key oncogenic pathway underlying neuroblastoma progression: specifically, MYCN, expressed at elevated level, transactivates the miRNA 17-5p-92 cluster, which inhibits p21 and BIM translation by interaction with their mRNA 3' UTRs. Overexpression of miRNA 17-5p-92 cluster in MYCN-not-amplified neuroblastoma cells strongly augments their in vitro and in vivo tumorigenesis. In vitro or in vivo treatment with antagomir-17-5p abolishes the growth of MYCN-amplified and therapy-resistant neuroblastoma through p21 and BIM upmodulation, leading to cell cycling blockade and activation of apoptosis, respectively. In primary neuroblastoma, the majority of cases show a rise of miR-17-5p level leading to p21 downmodulation, which is particularly severe in patients with MYCN amplification and poor prognosis. Altogether, our studies demonstrate for the first time that antagomir treatment can abolish tumor growth in vivo, specifically in therapy-resistant neuroblastoma.

Altered expression of endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 in transformed non-lymphoid human tissues.

The endoplasmic reticulum (ER) aminopeptidases ERAP1 and ERAP2 contribute to generate HLA class I binding peptides. Recently, we have shown that the expression of these enzymes is high and coordinated (with each other and with HLA class I molecules) in immortalized B cells, but variable and imbalanced in human tumour cell lines of various non-lymphoid lineages. Herein, this issue was investigated in vivo by testing ERAP1 and ERAP2 expression in normal non-lymphoid tissues and their malignant counterparts. ERAP1 and ERAP2 were detected exclusively in the epithelial cells of over half of the tested normal tissues. Four ERAP1/ERAP2 phenotypes (+/+, -/-, +/- and -/+) were detected, and the presence of either or both enzymes was not necessarily associated with HLA class I expression. In more than 160 neoplastic lesions, the expression of either or both aminopeptidases was retained, lost (most frequently, particularly ERAP1) or acquired as compared to the normal counterparts, depending on the tumour histotype. The double-negative (-/-) phenotype was the most frequent, and significantly (P = 0.013) associated with a lack of detectable HLA class I antigens. In selected neoplastic lesions, ERAP1 and ERAP2 were also tested for their enzymatic (peptide-trimming) activities. Expression and function were found to correlate, indicating that immunohistochemistry detects active enzymes in vivo. Thus, dissociation in the expression of ERAP1, ERAP2 and HLA class I may already be present in some normal tissues, but malignant transformation causes additional losses, gains and imbalances in specific tumour histotypes, and these alter the peptide-trimming ability of tumour cells in vivo.