Cindy Yen Okitsu

DNA Methylation Dictates Histone H3K4 Methylation

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.

Regionally Specific and Genome-Wide Analyses Conclusively Demonstrate the Absence of CpG Methylation in Human Mitochondrial DNA

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.

Transcriptional Activity Affects the H3K4me3 Level and Distribution in the Coding Region

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.

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.

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.

Sensitivity and specificity of immunoprecipitation of DNA containing 5-Methylcytosine

BackgroundAttempts 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.FindingsWe 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.ConclusionThe 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.