Construction of high coverage whole-genome sequencing libraries from single colon crypts without DNA extraction or whole-genome amplification.

OBJECTIVE: Comprehensive and reliable genome-wide variant analysis of a small number of cells has been challenging due to genome coverage bias, PCR over-cycling, and the requirement of expensive technologies. To comprehensively identify genome alterations in single colon crypts that reflect genome heterogeneity of stem cells, we developed a method to construct whole-genome sequencing libraries from single colon crypts without DNA extraction, whole-genome amplification, or increased PCR enrichment cycles. RESULTS: We present post-alignment statistics of 81 single-crypts (each contains four- to eight-fold less DNA than the requirement of conventional methods) and 16 bulk-tissue libraries to demonstrate the consistent success in obtaining reliable coverage, both in depth (≥ 30X) and breadth (≥ 92% of the genome covered at ≥ 10X depth), of the human genome. These single-crypt libraries are of comparable quality as libraries generated with the conventional method using high quality and quantities of purified DNA. Conceivably, our method can be applied to small biopsy samples from many tissues and can be combined with single cell targeted sequencing to comprehensively profile cancer genomes and their evolution. The broad potential application of this method offers expanded possibilities in cost-effectively examining genome heterogeneity in small numbers of cells at high resolution.

Temporally uncoupled signal and coding joint formation in human V(D)J recombination.

In vertebrate antigen receptor gene rearrangement, V(D)J recombination events can occur by deletion or by inversion. For deletional events, the signal joint is deleted from the genome. Nearly half of the immunoglobulin light chain genes undergo V(D)J recombination in an inversional manner, and both signal and coding joint formation must occur to retain chromosomal integrity. But given the undetermined amount of pre-B and pre-T cell death that occurs during V(D)J recombination, the efficiency with which both joints are completed is not known, nor is the relative efficiency (balance) of signal versus coding joint formation. Signal joint formation only requires Ku and XRCC4:DNA ligase 4 of the nonhomologous DNA end joining repair pathway. Coding joint formation requires these proteins as well, but in addition requires Artemis and DNA-dependent protein kinase to open the hairpin DNA coding ends, which the RAG complex generated; and further processing is required because the hairpin opening generates incompatible 3' overhangs. Mutations in some of the end processing enzymes affect one, but only minimally the other joint. We have devised a precise cellular assay that does not have any cellular, enzymatic or biochemical selective bias to assess signal and coding joint formation independently, and it can detect intermediates for which one joint has formed but not the other. We find that intermediates with only one completed joint are more abundant than molecules with both joints completed. This indicates that either joint can form independent of the other and joint formation can be a relatively slow process.

DNA-PKcs chemical inhibition versus genetic mutation: Impact on the junctional repair steps of V(D)J recombination.

Spontaneous DNA-PKcs deficiencies in animals result in a severe combined immunodeficiency (SCID) phenotype because DNA-PKcs is required to activate Artemis for V(D)J recombination coding end hairpin opening. The impact on signal joint formation in these spontaneous mutant mammals is variable. Genetically engineered DNA-PKcs null mice and cells from them show a >1,000-fold reduction in coding joint formation and minimal reduction in signal joint formation during V(D)J recombination. Does chemical inhibition of DNA-PKcs mimic this phenotype? M3814 (also known as Nedisertib) is a potent DNA-PKcs inhibitor. We find here that M3814 causes a quantitative reduction in coding joint formation relative to signal joint formation. The sequences of signal and coding junctions were within normal limits, though rare coding joints showed novel features. The signal junctions generally did not show evidence of resection into the signal ends that is often seen in cells that have genetic defects in DNA-PKcs. Comparison of the chemical inhibition findings here with the known results for spontaneous and engineered DNA-PKcs mutant mammals is informative for considering pharmacologic small molecule inhibition of DNA-PKcs in various types of neoplasia.

Sensitivity and specificity of immunoprecipitation of DNA containing 5-Methylcytosine.

BACKGROUND: Attempts to enrich or identify DNA with cytosine methylation have been commonly carried out using anti-5-methylcytosine or anti-MBD2 (methyl-CpG binding domain protein 2) antibody in immunoprecipitation (IP) assays. However, a careful and systematic control experiment to examine the sensitivity and specificity of this approach has not been reported. It is of critical importance to understand the potential pitfalls of this approach and to avoid potential misinterpretation of findings. FINDINGS: We found that increased concentration of antibody used in the assay increased the amount of overall DNA captured as expected. The increased number of methylated cytosines in/on the DNA fragment also increased the amount of DNA captured by the antibody. Importantly, the antibody can bind to some fully unmethylated DNA fragments, even when fully methylated DNA is present in the same experiment. CONCLUSION: The sensitivity of anti-5-methylcytosine antibody and anti-MBD2 antibody/MBD2 binding varies with the number of methylated cytosines on the DNA target. The specificity of these antibodies can also vary for different DNA target sequences. DNA fragments with fewer CpG sites may not bind to these antibodies even when all are methylated while DNA fragments with more CpG sites may bind to the antibodies when only some of these sites are methylated. More importantly, binding of DNA to these antibodies does not always indicate the presence of DNA methylation. It is clear that false positive and false negative findings can be easily reached even though it does not nullify these convenient and simple methods completely. Great caution should be taken for the interpretation of IP results using these antibodies and rigorous confirmation by sodium bisulfite sequencing is essential.

Effect of CpG dinucleotides within IgH switch region repeats on immunoglobulin class switch recombination.

Immunoglobulin (Ig) heavy chains undergo class switch recombination (CSR) to change the heavy chain isotype from IgM to IgG, A or E. The switch regions are several kilobases long, repetitive, and G-rich on the nontemplate strand. They are also relatively depleted of CpG (also called CG) sites for unknown reasons. Here we use synthetic switch regions at the IgH switch alpha (Sα) locus to test the effect of CpG sites and to try to understand why the IgH switch sequences evolved to be relatively depleted of CpG. We find that even just two CpG sites within an 80 bp synthetic switch repeat iterated 15 times (total switch region length of 1200 bp containing 30 CpG sites) are sufficient to dramatically reduce both Ig CSR and transcription through the switch region from the upstream Iα sterile transcript promoter, which is the promoter that directs transcripts through the Sα region. De novo DNA methylation occurs at the four CpG sites in and around the Iα promoter when each 80 bp Iα switch repeat contains the two CpG sites. Thus, a relatively low density of CpG sites within the switch repeats can induce upstream CpG methylation at the IgH alpha locus, and cause a substantial decrease in transcription from the sterile transcript promoter. This effect is likely the reason that switch regions evolved to contain very few CpG sites. We discuss these findings as they relate to DNA methylation and to Ig CSR.

Reproducibility and reliability of SNP analysis using human cellular DNA at or near nanogram levels.

BACKGROUND: Illumina SNP arrays have been routinely used for genome-wide association studies to identify potential biomarkers for various diseases. The recommended 200 ng of DNA for high-quality results is a roadblock to utilizing this assay when such quantities of DNA are not available. The goal of this study is to determine the reproducibility and reliability of the assay when reduced amounts of DNA are used for the SNP arrays. FINDINGS: A serial 3-fold reduction of DNA from 200 ng to 0.8 ng was used for an Illumina SNP array in duplicates (200 ng, 66.7 ng, 22,2 ng, and 7.4 ng) or triplicates (2.47 ng and 0.8 ng). The reproducibility of the assay was determined by comparing allele calls (genotypes) at each locus within the duplicates or triplicates. The reliability of samples of reduced quantity was determined by comparing allele calls from samples of different quantities. As expected, the reproducibility and reliability both decrease with decreasing amounts of DNA used for the arrays. However, results of comparable quality to the 200 ng DNA recommended by Illumina can be obtained with much reduced amounts of DNA. CONCLUSION: Reasonably reproducible and reliable results can be obtained with quantities of DNA, as low as 0.8 ng (equivalent to 133 human cells), well below the manufacturer's recommendation. Results of nearly equal quality to that of using 200 ng DNA can be obtained with 22.2 ng of DNA reliably, and clearly acceptable data can be obtained using 7.4 ng of DNA for Illumina SNP arrays.

Regionally specific and genome-wide analyses conclusively demonstrate the absence of CpG methylation in human mitochondrial DNA.

Although CpG methylation clearly distributes genome-wide in vertebrate nuclear DNA, the state of methylation in the vertebrate mitochondrial genome has been unclear. Several recent reports using immunoprecipitation, mass spectrometry, and enzyme-linked immunosorbent assay methods concluded that human mitochondrial DNA (mtDNA) has much more than the 2 to 5% CpG methylation previously estimated. However, these methods do not provide information as to the sites or frequency of methylation at each CpG site. Here, we have used the more definitive bisulfite genomic sequencing method to examine CpG methylation in HCT116 human cells and primary human cells to independently answer these two questions. We found no evidence of CpG methylation at a biologically significant level in these regions of the human mitochondrial genome. Furthermore, unbiased next-generation sequencing of sodium bisulfite treated total DNA from HCT116 cells and analysis of genome-wide sodium bisulfite sequencing data sets from several other DNA sources confirmed this absence of CpG methylation in mtDNA. Based on our findings using regionally specific and genome-wide approaches with multiple human cell sources, we can definitively conclude that CpG methylation is absent in mtDNA. It is highly unlikely that CpG methylation plays any role in direct control of mitochondrial function.

Heterogeneity and randomness of DNA methylation patterns in human embryonic stem cells.

DNA methylation has been proposed to be important in many biological processes and is the subject of intense study. Traditional bisulfite genomic sequencing allows detailed high-resolution methylation pattern analysis of each molecule with haplotype information across a few hundred bases at each locus, but lacks the capacity to gather voluminous data. Although recent technological developments are aimed at assessing DNA methylation patterns in a high-throughput manner across the genome, the haplotype information cannot be accurately assembled when the sequencing reads are short or when each hybridization target only includes one or two cytosine-phosphate-guanine (CpG) sites. Whether a distinct and nonrandom DNA methylation pattern is present at a given locus is difficult to discern without the haplotype information, and the DNA methylation patterns are much less apparent because the data are often obtained only as methylation frequencies at each CpG site with some of these methods. It would facilitate the interpretation of data obtained from high-throughput bisulfite sequencing if the loci with nonrandom DNA methylation patterns could be distinguished from those that are randomly methylated. In this study, we carried out traditional genomic bisulfite sequencing using the normal diploid human embryonic stem (hES) cell lines, and utilized Hamming distance analysis to evaluate the existence of a distinct and nonrandom DNA methylation pattern at each locus studied. Our findings suggest that Hamming distance is a simple, quick, and useful tool to identify loci with nonrandom DNA methylation patterns and may be utilized to discern links between biological changes and DNA methylation patterns in the high-throughput bisulfite sequencing data sets.

Q-PCR in combination with ChIP assays to detect changes in chromatin acetylation.

Quantitative polymerase chain reaction (Q-PCR) allows for the accurate and reproducible determination of the amount of target DNA in a sample through the measurement of PCR product accumulation in "real time." This method determines starting target DNA quantity over a large assay dynamic range and requires no post-PCR sample manipulation. When used in combination with the method of chromatin immunoprecipitation (ChIP), the amount of protein binding to a specific region of DNA can be accurately and rapidly determined. A method for quantifying the presence of acetylated histones H3 and H4 on different regions of a target locus using Q-PCR after ChIP is described.

Transcriptional activity affects the H3K4me3 level and distribution in the coding region.

Histone lysine methylation and CpG DNA methylation contribute to transcriptional regulation. We have shown previously that dimethylated and trimethylated forms of histone H3 at lysine 4 (H3K4me2 and H3K4me3) are primarily depleted from CpG-methylated DNA regions by using patch-methylated stable episomes (minichromosomes) in human cells. This effect on H3K4me2 is clearly not linked to the transcriptional activity in the methylated DNA region; however, transcriptional activity may play a role in the presence of H3K4me3. Here, we present clear evidence of the impact of transcriptional activity on the overall level of H3K4me3 in the coding region and the lack of impact on H3K4me2. Our data also demonstrate the influence of transcriptional activity on the distribution of H3K4me3 and H3K4me2, but not that of total H3, in the 5' end of the coding region relative to the 3' end. The nature of the promoter (viral or endogenous) affects H3K4me3 much more than it affects H3K4me2, suggesting a potential fundamental difference in the recruitment of methyltransferase for H3K4 trimethylation.

A simple and inexpensive on-column frit fabrication method for fused-silica capillaries for increased capacity and versatility in LC-MS/MS applications.

A modified sol-gel method for a one-step on-column frit preparation for fused-silica capillaries and its utility for peptide separation in LC-MS/MS is described. This method is inexpensive, reproducible, and does not require specialized equipments. Because the frit fabrication process does not damage polyimide coating, the frit-fabricated column can be tightly connected on-line for high pressure LC. These columns can replace any capillary liquid transfer tubing without any specialized connections up-stream of a spray tip column. Therefore multiple columns with different phases can be connected in series for one- or multiple-dimensional chromatography.

DNA methylation dictates histone H3K4 methylation.

Histone lysine methylation and DNA methylation contribute to transcriptional regulation. We have previously shown that acetylated histones are associated with unmethylated DNA and are nearly absent from the methylated DNA regions by using patch-methylated stable episomes in human cells. The present study further demonstrates that DNA methylation immediately downstream from the transcription start site has a dramatic impact on transcription and that DNA methylation has a larger effect on transcription elongation than on initiation. We also show that dimethylated histone H3 at lysine 4 (H3K4me2) is depleted from regions with DNA methylation and that this effect is not linked to the transcriptional activity in the region. This effect is a local one and does not extend even 200 bp from the methylated DNA regions. Although depleted primarily from the methylated DNA regions, the presence of trimethylated histone H3 at lysine 4 (H3K4me3) may be affected by transcriptional activity as well. The data here suggest that DNA methylation at the junction of transcription initiation and elongation is most critical in transcription suppression and that this effect is mechanistically mediated through chromatin structure. The data also strongly support the model in which DNA methylation and not transcriptional activity dictates a closed chromatin structure, which excludes H3K4me2 and H3K4me3 in the region, as one of the pathways that safeguards the silent state of genes.

Tumor necrosis factor alpha-dependent drug resistance to purine and pyrimidine analogues in human colon tumor cells mediated through IKK.

Development of drug resistance in cancer is one of the main challenges in chemotherapy, and many mechanisms are still unknown. In this study, we show that tumor necrosis factor alpha (TNFalpha) increases postdrug survival from 5-fluoro-2'-deoxyuridine (FdUrd) in two human colon tumor cell lines. This resulted in the development of drug-resistant cells in a TNFalpha-dependent manner. Interestingly, although the drug-resistant cells were selected using FdUrd, they are also resistant to a number of other antimetabolites in the DNA synthesis pathway in a TNFalpha-dependent manner. Only in the drug-resistant cells (p35-colo201) TNFalpha treatment resulted in G(0)-G(1) arrest but not in the parental colo201 and other cell types. Blocking TNFalpha-induced cell cycle arrest sensitized drug-resistant cells to FdUrd. TNFalpha-induced cell cycle arrest required IKK. IKK inhibition by a small molecule inhibitor or by the knockdown of IKKalpha, IKKbeta, or RelA/p65 using siRNA, but not the inhibition of JNK, MEK, p38, or caspase-8 pathways, blocked TNFalpha-induced G(0)-G(1) arrest and restored sensitivity to FdUrd of drug-resistant cells. TNFalpha reduced the transcripts and protein levels of phosphorylated retinoblastoma protein (Rb), Rb, E2F1, and Cdk4 only in drug-resistant p35-colo201 cells. This effect of TNFalpha was reversed by IKK inhibitor, suggesting that TNFalpha-induced cell cycle arrest is probably due to the reduction of Rb, E2F1, and Cdk4. Taken together, this study shows that, in vitro, TNFalpha-induced cell cycle arrest through IKK can provide a mechanism for the development of drug resistance to anti-cancer drugs, purine and pyrimidine analogues.