Genomic imprinting and X-chromosome inactivation are well-described phenomena in which allele-specific gene expression occurs, although the underlying molecular mechanisms for this remain incompletely understood. A number of recent studies have demonstrated that allele-specific gene expression also occurs among autosomal, nonimprinted genes (1-4). Allele-specific effects appear to be heritable, of relatively modest magnitude (typically 1.5 to 2-fold), highly context specific, and occur relatively commonly. Moreover, differences in gene expression can be mapped as quantitative traits (4). There is growing interest in the phenomenon of allele-specific differences both as evidence to support the existence of cis-acting regulatory polymorphisms of the DNA sequence and as a tool for the identification of regulatory DNA variation (reviewed in

From: Methods in Molecular Biology, vol. 338: Gene Mapping, Discovery, and Expression: Methods and Protocols Edited by: M. Bina © Humana Press Inc., Totowa, NJ

refs. 5-7). At present the identification of genetic variation modulating gene expression remains highly problematic (8). This is important, as regulatory polymorphisms account for a significant amount of interindividual variation in gene expression (6) and are likely to underlie many of the observed associations between genetic variation and susceptibility to disease in population-based genetic association studies (9).

In vitro strategies for allele-specific analysis of gene expression provide important experimental evidence of the effects of DNA sequence diversity. These have typically involved reporter gene analysis and assays of protein-DNA binding (10). In both cases, polymorphisms can be engineered into the DNA sequence and then either incorporated into a reporter gene construct for trans-fection into a cell of interest or used as probes to compare relative protein-DNA binding affinity. This chapter describes approaches that analyze allele-specific gene expression in living cells. Here it is the naturally occurring sequence diversity that is analyzed in a context of the normal chromatin and regulatory mechanisms controlling gene expression.

To achieve this, the analysis of allele-specific gene expression in vivo of autosomal genes has relied on defining the allelic origin of RNA using a transcribed marker polymorphism and quantifying the relative allele-specific transcript abundance (1,11). If a DNA marker such as a single-nucleotide polymorphism (SNP) is selected in a heterozygous state, with one copy of each allele within the cell, this provides an internally controlled situation in which the relative abundance of the transcript can be assayed (Fig. 1). This contrasts with the situation of correlating either total RNA abundance or the resulting translated protein between cells or individuals on the basis of their genotype. Such analyses are potentially confounded at many levels, such as prevailing environmental stimuli, variation in the signaling cascade, and the genetic makeup of the individual.

Chromatin immunoprecipitation affords a further insight into allele-specific effects on gene expression, as proteins bound to DNA can be quantified in an allele-specific manner, again discriminated on the basis of a marker polymorphism but here assaying DNA rather than RNA and thus circumventing the need for the polymorphism to be transcribed. Allele- or haplotype-specific chromatin immunoprecipitation (haploChIP) allows for both discrimination of allele-specific transcription factor binding (12) and Pol II loading (13). The latter, when assayed using antibodies to phosphorylated Pol II, may serve as a useful surrogate of levels of gene expression, expanding the number of polymorphisms and haplotypes that can be analyzed for allele-specific effects (Fig. 1).

A number of different approaches have been used to quantify the relative allelic abundance of RNA (in allele-specific transcript analysis) or DNA (in allele-specific ChIP), notably primer extension assays. A number of modes of

Fig. 1. Allelic discrimination using heterozygous polymorphic markers. In a cell in which there is a relative allelic difference in gene expression seen in transcript abundance, this may arise owing to ds-acting regulatory polymorphisms. For example, SNP 1 may act to change a site of DNA-protein binding resulting in recruitment of a transcriptional repressor to one allele. In the presence of a transcribed marker (SNP 3), the relative abundance of RNA can be determined according to its allelic origin. Phosphory-lated Pol II loading can also be used to estimate relative gene expression using haplo-ChlP, but here any polymorphic marker within 1 kb of the 5' or 3' end of the gene can be used, as the method does not rely on the marker being transcribed: in this case SNPs 1, 2, or 3 can be used. HaploChIP can also be used to estimate relative allele-specific binding by the transcriptional repressor using SNPs 1, 2, or 3 (if the SNPs lie within 1 kb of the transcriptional repressor binding site).

quantification have been reported with detection using radionucleotides (11), fluorescence (1,14), and mass spectrometry (13). Other strategic approaches to defining relative allelic abundance include the amplification refractory mutation system (ARMS) (15) and polymerase chain reaction (PCR) amplification with restriction fragment-length polymorphism analysis. The incorporation of a labeled primer in the final amplification cycle of PCR amplification (hot-stop PCR) offers the advantage of avoiding heteroduplex formation, which may bias observed allelic differences (16). High-throughput approaches based on microarray technology have also been used (3) together with single-molecule RNA profiling (17), but because of space limitations these two approaches are not described here.

2. Materials

2.1. Choice of Cell Type and Ascertainment of Heterozygosity

1. PCR amplification: BioTaq DNA polymerase (Bioline, London, UK), DNA Engine thermal cycler (MJ Research, Waltham, MA).

2.2. Cell Culture and Harvesting

1. Culture medium for lymphoblastoid cell lines (LCLs): RPMI-1640 supplemented with 2 mM glutamine and 10% fetal bovine serum.

2. Phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, Gillingham, Dorset, UK) dissolved at 1 mM in dimethyl sulfoxide (DMSO) and stored at -20°C.

3. Ionomycin (Sigma) dissolved at 1 mM in DMSO and stored at -20°C.

4. TRIzol Reagent (Invitrogen, Paisley, UK).

5. Formaldehyde crosslinking buffer (10X): 100 mM NaCl, 1 mM EDTApH 8, 0.5, mM EGTA pH 8, 50 mM HEPES, pH 8. Autoclave and store at room temperature.

6. 11% Formaldehyde (10X) to be added to 10X formaldehyde crosslinking buffer immediately prior to use in a fume hood.

7. Glycine solution (20X, 2.5 M): autoclave and store at room temperature.

8. Phosphate-buffered saline (PBS, 1X): autoclave and store at 4°C.

2.3. Extraction of RNA and cDNA Synthesis

1. RNase-free DNase I (Ambion [Europe], Huntingdon, Cambridgeshire, UK).

2. AMV reverse transcriptase (Roche Diagnostics, Lewes, East Sussex, UK).

3. RNasein Ribonuclease Inhibitor (Promega, UK, Southampton, UK).

2.4. Chromatin Immunoprecipitation

1. ChIP lysis buffer 1 (1X): 50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40 (Calbiochem, San Diego, CA), 0.25% Triton X-100 (Sigma). Store at 4°C.

2. ChIP lysis buffer 2 (1X): 200 mM NaCl, 1 mM EDTA pH 8, 0.5 mM EGTA pH 8, 10 mM Tris-HCl, pH 8. Store at room temperature.

3. ChIP lysis buffer 3 (1X): 1 mM EDTA, pH 8, 0.5 mM EGTA, pH 8, 10 mM Tris-HCl, pH 8. Store at 4°C.

4. ChIP lysis buffers 1, 2, and 3 should be supplemented with protease inhibitors immediately before use: complete protease inhibitor tablets (Roche), 1 mM benzamidine (Sigma; 0.1 M stock prepared with water and stored at -20°C), 50 ^g/mL TLCK (Roche) (1 mg/mL stock in 0.05 M sodium acetate, pH 5 [Sigma], stored at -20°C), 50 ^g/mL TPCK (Roche; stock 3 mg/mL in ethanol, stored at -20°C), 1 ^g/mL pep-statin (Roche; stock 1 mg/mL in ethanol, stored at -20°C).

5. Branson 450 Sonifier (Branson Ultrasonics, Branson, CT).

6. The choice of antibody will depend on the application; for analysis of gene expression, antibodies vs phosphorylated serine residues of the CTD of Pol II (Ser5, MMS-134R clone H14; Ser2, MMS-129R clone H5; Covance, Princeton, NJ) may be used.

7. Dynabeads M-280 (Dynal Biotech, Oslo, Norway) with secondary antibody of choice.

8. Magnetic particle concentrator (MPC; Dynal).

9. To wash Dynabeads, prepare a solution of bovine serum albumin (BSA) 5 mg/mL in PBS immediately before use.

10. Nutator (Shelton Scientific, Shelton, CT).

11. 2X RIPA-POL buffer: 20 mM Tris-HCl, pH 8, 2 mM EDTA, 1 mM EGTA, 2% Triton X-100 (Sigma), 0.2% sodium deoxycholate, 0.2% sodium dodecyl sulfate (SDS), 280 mM NaCl, 2X complete protease inhibitor, and 10 ^g/mL pepstatin. When required, add 100 ^g/mL sonicated herring sperm DNA (Promega, Madison, WI) and 250 mM lithium chloride.

12. Elution buffer: 10 mM Tris-HCl, pH 8, 1 mM EDTA, 1% SDS. Store at room temperature.

13. Qiagen DNA clean-up kit (Qiagen, Crawley, West Sussex).

2.5. Quantification of Relative Allelic Abundance

1. Primer extension quantification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS): massEXTEND platform (Sequenom, San Diego, CA) including SpectroCLEAN (Sequenom) resin, SpectroCHIP (Sequenom) microarray, SpectroPOINT (Sequenom) nanoliter dispenser, and a Spectro-READER (Sequenom) mass spectrometer.

2. SNaPshot Multiplex Kit (Applied Biosystems, Warrington, Cheshire, UK).

3. Methods

3.1. Choice of Cell Type and Ascertainment of Heterozygosity

1. Genomic DNA from the cells or tissue selected for analysis (see Note 1) is geno-typed using ARMS (see Note 2). This will define genetic markers (see Note 3), which can then be used to resolve the allelic origin of RNA or immunoprecipitated DNA as described in Subheading 3.5.

2. Primers are designed with a 3' mismatch at the site of the polymorphism and multiplexed with a control primer set (see Note 4). PCR amplification is performed using 1X PCR buffer, 0.4 mM dNTPs, 1.9 mM MgCl2, 0.25 U Bioline Taq. Amplification is performed at 96°C for 1 min; 5 cycles of 96°C for 35 s, 70°C for 45 s, 72°C for 35 s; 23 cycles of 96°C for 25 s, 65°C for 50 s, 72°C for 40 s; 6 cycles of 96°C for 35 s, 55°C for 1 min, 72°C for 1.5 min. Products are visualized by agarose gel electrophoresis.

3.2. Cell Culture and Harvesting

1. LCLs (see Note 5) are grown in RPMI-1640 medium supplemented with penicillin and streptomycin, with 2 mM L-glutamine and 10% heat-inactivated fetal bovine serum. Cells are harvested in mid log phase.

2. For RNA preparation, 10 to 50 o 106 cells are typically harvested for each time point of interest (see Note 6).

a. Cell suspensions are centrifuged at 500g for 5 min at 4°C and then placed on ice; culture media are removed by aspiration.

b. The cell pellet is lysed in TRIzol (see Note 7) by repetitive pipeting using 1 mL TRIzol per 10 o 106 cells.

c. The lysed material can be stored at this point at -80°C or the RNA isolated directly.

3. For chromatin preparation 100 to 500 o 106 cells are used per time point.

a. Crosslinking buffer (10X) containing formaldehyde is added directly to cells in growing media, gently mixed, and left to incubate for 45 min at room temperature (see Note 8).

b. Then 2.5 M glycine (20X) is added to stop crosslinking, and cells are pelleted by centrifugation at 500g for 5 min at 4°C.

c. The cells are washed in cold PBS and then either used directly to isolate nuclei or stored as a cell pellet at -80°C (see Note 9).

3.3. Extraction of RNA and cDNA Synthesis

1. RNA is isolated using TRIzol reagent according to the manufacturer's instructions.

a. Briefly, the homogenized sample is incubated for 5 min at room temperature and then 0.2 mL chloroform is added per 1 mL TRIzol reagent.

b. Following shaking by hand for 15 s and incubation at room temperature for 2 min, samples are centrifuged at 12,000g for 15 min at 4°C.

c. The colorless upper aqueous phase is transferred to a new tube and precipitated using isopropyl alcohol (0.5 mL per 1 mL TRIzol used for initial homogenization).

d. The sames are incubated at room temperature for 10 min and centrifuged at 12,000g for 10 min at 4°C.

e. Supernatant is removed, and the RNA pellet is washed with 75% ethanol.

f. Purified total RNA is then treated with DNase I (for a 50-^g aliquot of total RNA, 8 U DNase I for 40 min at 37°C) followed by phenol-chloroform extraction and reprecipitation (see Note 10).

2. The RNA is annealed to random decamers and first-strand cDNA synthesis is performed using AMV reverse transcriptase (see Note 11) incorporating appropriate negative controls.

a. The reaction mix comprises 2.5 ^g RNA in PCR buffer with 1 mM dNTP, 2.5 mM MgCl2, 10 U/sample RNasin, 5 U AMV RT, and 10 ^ M random decamer.

b. Reactions are incubated in a thermal cycler at 25°C for 10 min and then 42°C for 60 min and placed on ice before purification using a spin column.

3.4. Chromatin Immunoprecipitation

1. Nuclear material is prepared from cell pellets by resuspending cells on ice in 20 mL

ChIP lysis buffer 1 per 500 o 106 cells (see Note 12).

a. Rock suspension at 4°C for 10 min and then pellet in tabletop centrifuge at 2300g for 10 min at 4°C.

b. Resuspend pellet in 16 mL ChIP lysis buffer 2 (containing protease inhibitors) at room temperature, and rock gently at room temperature for 10 min.

c. Repeat centrifugation at 4°C, and resuspend the nuclear material on ice in 4 mL ChIP lysis buffer 3 (containing protease inhibitors).

2. The suspension of nuclear material is then sonicated to achive a final average DNA fragment size of 0.5 to 1 kb (see Note 13).

3. Sonicated material is centrifuged at 12,000g at 4°C for 10 min and adjusted to 10% final glycerol concentration prior to storage at -80°C or used directly in the immu-noprecipitation reactions described below (see Note 14) in steps 4 to 11.

4. Immunoprecipitation reactions are prepared by combining 50 ^g chromatin with 2 ^g primary antibody (such as vs phosphorylated Pol II) attached to 50 ^L starting volume of Dynabeads. The experimental design should include appropriate negative controls.

5. Dynabead-antibody complexes are prepared by concentrating Dynabeads M280 precoated with secondary antibody in an MPC, removing supernatant, and resus-pending Dynabeads in 1 mL PBS/BSA.

a. Beads are washed twice in PBS/PSA and primary antibody is added, followed by incubation overnight on a nutator at 4°C.

b. To remove unbound antibody, use MPC and wash in PBS/BSA three times.

c. Beads are resuspended in a volume of PBS/BSA equal to the starting volume of Dynabeads taken from stock.

6. To set up immunoprecipitation reactions, combine chromatin with Dynabeads bound to primary antibody to achieve a final concentration of 1X RIPA-POL in a total volume of 500 ^L. Incubate overnight on a nutator at 4°C.

7. Save aliquots of chromatin from antibody negative control tube to use as input control (typically take 2.5 ^L, make up to 100 ^L with TE, and store on ice) and then wash immunoprecipitation reactions using MPC (see Note 15).

a. Sequentially wash bead complexes twice with 1 mL freshly prepared 1X RIPA-POL buffer.

b. Proceed to wash with 1X RIPA-POL buffer containing 100 ^g/mL herring sperm DNA, 1X RIPA-POL buffer with 100 ^g/mL herring sperm DNA plus 300 mM NaCl, and finally 1X RIPA-Pol with 250 mM LiCl, rocking at room temperature for exactly 5 min with each wash.

c. Wash once with 1 mL TE, and remove any remaining liquid.

8. Elute from beads.

a. First, 50 ^L of elution buffer is added, vortexed briefly to resuspend beads, and incubated at 65°C for 10 min.

b. Samples are centrifuged for 30 s at maximum speed in a microfuge and supernatant is transfered to a new tube.

c. The remaining bead pellet is discarded.

d. Then 120 ^L of elution buffer is added to the supernatant in the new tube, and crosslinks are reversed by incubating at 65°C overnight in water bath.

e. For input controls, add 11 ^L of 10% SDS and reverse crosslink at 65°C overnight in water bath.

9. To remove proteins, 150 ^L of proteinase K/glycogen mix is added to each tube and incubated for 2 h at 37°C.

10. DNA extraction.

a. An equal volume of equilibrated phenol-chloroform, pH 8, is added and then vortexed and spun at maximum speed in a microfuge for 5 min.

b. Phenol-chloroform extraction is repeated and then DNA is extracted once with an equal volume of chloroform/isoamyl alcohol.

c. To precipitate DNA fragments, 1/10th vol of 3 Msodium acetate, pH 5.2, 2.5X volume of ice-cold 100% ethanol are added and vortexed briefly.

d. Samples are incubated at -20°C overnight and then centrifuged at maximum speed in a microfuge for 10 min at 4°C.

e. The resulting pellet is washed with 1 mL ice-cold 70% ethanol, vortexed, cen-trifuged for 5 min at 4°C at maximum speed, then air-dried.

11. To remove any contaminating RNA, the pellet is resuspended in 30 ^L TE containing 10 ^g RNase A and incubated for 1 h at 37°C. RNase can be removed by spin column purification according to the manufacturer's instructions and the DNA fragments eluted in 10 mM Tris-HCl, pH 8, and stored at -20°C.

3.5. Quantification of Relative Allelic Abundance

1. Non-allele-specific analysis should first be performed to check the specificity and abundance of the gene of interest using either RNA or the products of chromatin immunoprecipitation. Gene-specific PCR primers can be designed for analysis either as a uniplex or by multiplexing with a housekeeping gene to carry out semiquantitative PCR (18) or by quantitative PCR. It is important to include the negative controls (such as AMV negative samples from RT-PCR or mock antibody controls from ChIP) in this analysis.

2. Allele-specific quantification can be achieved by many approaches; primer extension has been most commonly used. A number of approaches are described below in steps 3 to 6, as many laboratories may not have the equipment to allow application of techniques such as fluorescent nucleotide detection or MALDI-TOF MS (see Note 16).

3. For primer extension with detection by mass spectrometry (PE/MS), an appropriate experimental design to minimize variance should be considered (see Note 17).

a. A first-round PCR reaction is performed using approx 1/100th of the product of the RT-PCR reaction (see Subheading 3.3.2.) or 1/25th of the ChIP DNA (see Subheading 3.4.11.) in a 25 ^L reaction vol using 0.5 U BioTaq with 0.8 mM dNTPs, 1.9 mM MgCl2, and 0.2 ^ M each primer.

b. Thermal cycling parameters should be optimized to ensure that the cycle number remains in the linear phase of amplification with annealing temperatures dependent on the primer design, for example, 96°C for 1 min followed by 6 cycles of 94°C for 45 s, 56°C for 45 s, 72°C for 30 s; then 30 cycles of 94°C for 45 s, 65°C for 45 s, 72°C for 30 s; followed by final extension at 72°C for 10 min.

c. The PCR product is then subaliquoted onto a 384-well plate, and nonincorpo-rated dNTPs are removed using shrimp alkaline phosphatase by incubating at 37°C for 20 min followed by 85°C for 5 min.

d. Primer extension is performed using a homogeneous MassEXTEND reaction comprising a cocktail of 100 ^M extension primer, 0.576 U MassEXTEND enzyme, buffer, and an appropriate deoxy and dideoxy nucleotide termination mix.

e. A typical primer extension reaction would comprise 94°C for 2 min then 40 cycles of 94°C for 5 s, 52°C for 5 s, 72°C for 5 s.

f. The products of primer extension are desalted using SpectroCLEAN resin and transferred onto a microarray by SpectroPOINT nanolitre dispenser.

g. MALDI-TOF analysis is performed using a SpectroREADER mass spectrometer.

4. Primer extension analysis by fluorescence is based on the single-base extension of an unlabeled oligonucleotide primer at its 3' end with a fluorescently labeled dideoxyterminator followed by quantification using capillary gel electrophoresis.

a. PCR-amplified samples are incubated with shrimp alkaline phosphatase and exonuclease I and then primer extension is performed using the SNaPshot Multiplex kit according to the manufacturer's instructions (see Note 18).

b. Primer extension cycling conditions for extension primers between 20 and 22 bp are 95°C for 2 min and then 25 cycles of 95°C for 5 s, 43°C for 5 s, 60°C for 5 s.

c. Products are treated with 0.5 U shrimp alkaline phosphatase for 45 min at 37°C and then at 85°C for 15 min.

d. The SNaPshot reaction is analyzed by electrophoresis on an ABI PRISM 310 or 3100 Genetic Analyzer or 3700 DNA Analyzer.

5. With Allele-specific quantification by hot-stop PCR, it is possible to resolve relative allelic abundance by PCR amplification followed by restriction enzyme digestion if the marker polymorphism creates a convenient restriction enzyme site. The problem with this and other PCR-based approaches is that heteroduplexes may occur and confound results. For this reason, hot-stop PCR can be a useful approach (16).

a. cDNA or the products of chromatin immunoprecipitation are amplified by PCR in 1X PCR buffer, 0.4 mM dNTPs, 1.5 mM MgCl2, 1 ^M each primer spanning the region of interest containing the marker polymorphism and Taq (94°C for 2 min then 30 cycles of 94°C for 30 s, 60°C for 15 s, 72°C for 20 s followed by 72°C for 10 min).

b. A final cycle using a labeled primer is now performed.

c. The chosen primer is radiolabeled with [y-32P]ATP using T4 PNK, and unincorporated radioactivity is removed by spin column.

d. A final round of PCR is performed following addition of 1 pmol of radiolabeled primer (94°C 30 s, 60°C 30 s, 72°C for 10 min).

e. The PCR product is digested using a restriction enzyme of choice and visualized by denaturing polyacrylamide gel electrophoresis.

6. Allele-specific quantification by the amplification refractory mutation system (ARMS) provides a simple and robust approach, although it may not be as the quantitative as the other approaches outlined above. The technique is described in Subheading 3.1. (see Note 19).

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