[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Background
Long genomic R-loops in eukaryotes were first described at the immunoglobulin heavy chain locus switch regions using bisulfite sequencing and functional studies. A mouse monoclonal antibody called S9.6 has been used for immunoprecipitation (IP) to identify R-loops, based on the assumption that it is specific for RNA:DNA over other nucleic acid duplexes. However, recent work has demonstrated that a variable domain of S9.6 binds AU-rich RNA:RNA duplexes with a KD that is only 5.6-fold weaker than for RNA:DNA duplexes. Most IP protocols do not pre-clear the genomic nucleic acid with RNase A to remove free RNA. Fold back of ssRNA can readily generate RNA:RNA duplexes that may bind the S9.6 antibody, and adventitious binding of RNA may also create short RNA:DNA regions. Here we investigate whether RNase A is needed to obtain reliable IP with S9.6.
As our test locus, we chose the most well-documented site for kilobase-long mammalian genomic R-loops, the immunoglobulin heavy chain locus (IgH) class switch regions. The R-loops at this locus can be induced by using cytokines to stimulate transcription from germline transcript promoters. We tested IP using S9.6 with and without various RNase treatments. The RNase treatments included RNase H to destroy the RNA in an RNA:DNA duplex and RNase A to destroy single-stranded (ss) RNA to prevent it from binding S9.6 directly (as duplex RNA) and to prevent the ssRNA from annealing to the genome, resulting in adventitious RNA:DNA hybrids. We find that optimal detection of RNA:DNA duplexes requires removal of ssRNA using RNase A. Without RNase A treatment, known regions of R-loop formation containing RNA:DNA duplexes can not be reliably detected. With RNase A treatment, a signal can be detected over background, but only within a limited 2 or 3-fold range, even with a stable kilobase-long genomic R-loop.
Any use of the S9.6 antibody must be preceded by RNase A treatment to remove free ssRNA that may compete for the S9.6 binding by forming RNA:RNA regions or short, transient RNA:DNA duplexes. Caution should be used when interpreting S9.6 data, and confirmation by independent structural and functional methods is essential.
Electronic supplementary material
The online version of this article (doi:10.1186/s13104-015-1092-1) contains supplementary material, which is available to authorized users.
Full-text · Article · Apr 2015 · BMC Research Notes
[Show abstract][Hide abstract] ABSTRACT: The boundaries of R-loops are well-documented at immunoglobulin heavy chain loci in mammalian B cells. Within primary B cells
or B cell lines, the upstream boundaries of R-loops typically begin early in the repetitive portion of the switch regions.
Most R-loops terminate within the switch repetitive zone, but the remainder can extend a few hundred base pairs further, where
G-density on the non-template DNA strand gradually drops to the genome average. Whether the G-density determines how far the
R-loops extend is an important question. We previously studied the role of G-clusters in initiating R-loop formation, but
we did not examine the role of G-density in permitting the elongation of the R-loop, after it had initiated. Here, we vary
the G-density of different portions of the switch region in a murine B cell line. We find that both class switch recombination
(CSR) and R-loop formation decrease significantly when the overall G-density is reduced from 46% to 29%. Short 50 bp insertions
with low G-density within switch regions do not appear to affect either CSR or R-loop elongation, whereas a longer (150 bp)
insertion impairs both. These results demonstrate that G-density is an important determinant of the length over which mammalian
genomic R-loops extend.
Full-text · Article · Nov 2014 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: R loops exist at the murine IgH switch regions and possibly other locations, but their functional importance is unclear. In biochemical systems, R loop initiation requires DNA sequence regions containing clusters of G nucleotides, but cellular studies have not been done. Here, we vary the G-clustering, total switch region length, and the number of target sites (WGCW sites for the activation-induced deaminase) at synthetic switch regions in a murine B cell line to determine the effect on class switch recombination (CSR). G-clusters increase CSR regardless of their immediate proximity to the WGCW sites. This increase is accompanied by an increase in R loop formation. CSR efficiency correlates better with the absolute number of WGCW sites in the switch region rather than the total switch region length or density of WGCW sites. Thus, the overall strength of the switch region depends on G-clusters, which initiate R loop formation, and on the number of WGCW sites.
[Show abstract][Hide abstract] ABSTRACT: Of the three DNA ligases present in all vertebrates, DNA ligase I (Lig1) has been considered essential for ligating Okazaki fragments during DNA replication and thereby essential for cell viability. Here, we report the striking finding that a Lig1-null murine B cell line is viable. Surprisingly, the Lig1-null cells exhibit normal proliferation and normal immunoglobulin heavy chain class switch recombination and are not hypersensitive to a wide variety of DNA damaging agents. These findings demonstrate that Lig1 is not absolutely required for cellular DNA replication and repair and that either Lig3 or Lig4 can substitute for the role of Lig1 in joining Okazaki fragments. The establishment of a Lig1-null cell line will greatly facilitate the characterization of DNA ligase function in mammalian cells, but the finding alone profoundly reprioritizes the role of ligase I in DNA replication, repair, and recombination.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · May 2013 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Little is known about the types and numbers of mutations that may accumulate in normal human cells with age. Such information would require obtaining enough DNA from a single cell to accurately carry out reliable analysis with extensive amplification, and complete genomic coverage under these circumstances is difficult. We have compared colon crypts, which are putatively clonal and contain ~2000 cells each, to determine how much somatic genetic variation occurs in vivo (without ex vivo cell culturing). Using high density SNP microarrays, we find that chromosome deletions, duplications and gene conversions were significantly more frequent in colons from the older individuals. These changes affected lengths ranging from 73kb to 46 Mb. Although detection requires progeny of a single mutant stem cell to reach niche dominance over neighboring stem cells, none of the deletions appear likely to confer a selective advantage. Mutations can become fixed randomly during stem cell evolution through neutral drift in normal human crypts. The fact that chromosomal changes are detected in individual crypts with increasing age suggests that either such changes accumulate with age or single stem cell dominance increases with age, and the former is more likely. This progressive genome-wide divergence of human somatic cells with age has implications for aging and disease in multicellular organisms.
[Show abstract][Hide abstract] ABSTRACT: The t(14;18) chromosomal translocation typically involves breakage at the bcl-2 major breakpoint region (MBR) to cause human follicular lymphoma. A theory to explain the striking propensity of the MBR
breaks at three CpG clusters within the 175-bp MBR region invoked activation-induced deaminase (AID). In a test of that theory,
we used here minichromosomal substrates in human pre-B cell lines. Consistent with the essential elements of the theory, we
found that the MBR breakage process is indeed highly dependent on DNA methylation at the CpG sites and highly dependent on
the AID enzyme to create lesions at peak locations within the MBR. Interestingly, breakage of the phosphodiester bonds at
the AID-initiated MBR lesions is RAG dependent, but, unexpectedly, most are also dependent on Artemis. We found that Artemis
is capable of nicking small heteroduplex structures and is even able to nick single-base mismatches. This raises the possibility
that activated Artemis, derived from the unjoined D to JH DNA ends at the IgH locus on chromosome 14, nicks AID-generated TG mismatches at methyl CpG sites, and this would explain
why the breaks at the chromosome 18 MBR occur within the same time window as those on chromosome 14.
Full-text · Article · Dec 2012 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jan 2012 · DNA and cell biology
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jan 2011 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Jun 2010 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Upon transcription of some sequences by RNA polymerases in vitro or in vivo, the RNA transcript can thread back onto the template
DNA strand, resulting in an R loop. Previously, we showed that initiation of R-loop formation at an R-loop initiation zone
(RIZ) is favored by G clusters. Here, using a purified in vitro system with T7 RNA polymerase, we show that increased distance
between the promoter and the R-loop-supporting G-rich region reduces R-loop formation. When the G-rich portion of the RNA
transcript is downstream from the 5′ end of the transcript, the ability of this portion of the transcript to anneal to the
template DNA strand is reduced. When we nucleolytically resect the beginning of the transcript, R-loop formation increases
because the G-rich portion of the RNA is now closer to the 5′ end of the transcript. Short G-clustered regions can act as
RIZs and reduce the distance-induced suppression of R-loop formation. Supercoiled DNA is known to favor transient separation
of the two DNA strands, and we find that this favors R-loop formation even in non-G-rich regions. Most strikingly, a nick
can serve as a strong RIZ, even in regions with no G richness. This has important implications for class switch recombination
and somatic hypermutation and possibly for other biological processes in transcribed regions.
Full-text · Article · Oct 2009 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: We have assembled, annotated, and analyzed a database of over 1700 breakpoints from the most common chromosomal rearrangements in human leukemias and lymphomas. Using this database, we show that although the CpG dinucleotide constitutes only 1% of the human genome, it accounts for 40%-70% of breakpoints at pro-B/pre-B stage translocation regions-specifically, those near the bcl-2, bcl-1, and E2A genes. We do not observe CpG hotspots in rearrangements involving lymphoid-myeloid progenitors, mature B cells, or T cells. The stage specificity, lineage specificity, CpG targeting, and unique breakpoint distributions at these cluster regions may be explained by a lesion-specific double-strand breakage mechanism involving the RAG complex acting at AID-deaminated methyl-CpGs.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class
switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for
this targeting. Here, we show that the difference between various forms of the Sμ locus that can or cannot undergo CSR correlates
well with the locations and detectability of R-loops. The Sμ R-loops can initiate hundreds of base pairs upstream of the core
repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins
to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of
the presence of the core Sμ repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These
findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core Sμ repeats but the marked
reduction in CSR in mice with deletions of the region upstream of the core Sμ repeats. These studies also provide the first
analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence.
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · May 2007 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: A role for pro-inflammatory cytokines in inflammation-related cancers has been suggested, but mechanisms are not defined. Here, we demonstrate that treatment of HeLa cells with TNFalpha increases chromosomal aberration. In contrast, IL-1beta did not increase, but rather decreased chromosomal aberration. TNFalpha and IL-1beta increased the production of H2O2 to similar levels in cells, suggesting that increased production of reactive oxygen species might not be the premier factor involved. Reducing H2O2 through overexpression of catalase or treatment of cells with NAC or BHA did not have an effect on TNF-induced chromosomal aberration. TNFalpha-induced NO production has been implicated in DNA damage. Inhibiting NO did not reduce TNF-induced chromosomal aberration. Inhibiting IKK, JNK, and p38 kinase as well as caspases decreased TNF-induced chromosomal aberration, and a correlation between TNF-induced apoptosis and CA generation was not found. Single-strand DNA breaks give rise to double-strand breaks, which then results in chromosomal breaks, when replication forks reach the single-strand breaks during S-phase. In cells progressing through S-phase, TNFalpha activation of IKK, JNK, and p38 is significantly reduced. However, these kinases were activated by IL-1beta in S-phase. The possibility that these pathways, in a TNF-specific manner, may regulate either the generation of single- and double-strand breaks or their repair, thereby resulting in increased chromosomal aberration, is discussed.