Retention of the Native Epigenome in Purified Mammalian Chromatin

<div><p>A protocol is presented for the isolation of native mammalian chromatin as fibers of 25–250 nucleosomes under conditions that preserve the natural epigenetic signature. The material is composed almost exclusively of histones and DNA and conforms to the structure expected by electron microscopy. All sequences probed for were retained, indicating that the material is representative of the majority of the genome. DNA methylation marks and histone marks resembled the patterns observed in vivo. Importantly, nucleosome positions also remained largely unchanged, except on CpG islands, where nucleosomes were found to be unstable. The technical challenges of reconstituting biochemical reactions with native mammalian chromatin are discussed.</p></div

Loss of GC-rich nucleosomes during purification.

<p>(A) Normalized read counts for nucleosomes extracted from nuclei or from purified genomic chromatin digested to mononucleosomes, averaged around all TSS’s. Note greater depletion around the TSS in the purified material than in the nuclei. (B) Nucleosome loss after purification as a function of nucleosomal GC content. Nucleosome read counts from nuclei and purified chromatin were counted in 500 bp windows across the genome. The log2 ratio of the two is displayed. The grey line shows a fitted Loess function, and the dashed line shows the average genomic GC content of 41%. (C) Normalized read counts averaged around all CpG islands (CGI).</p

Mass spectrometry analysis of genomic chromatin.

<p>Mass spectrometry analysis of genomic chromatin.</p

Nucleosome positions.

<p>Nucleosome positions on the promoters of one active locus (<i>Gapdh</i>) and two repressed loci (<i>Nanog</i> and <i>Pou5f1</i>) were mapped by ChIP-qPCR of mononucleosomes from purified genomic chromatin (black) and from nuclei (red). Peak height reflects nucleosome occupancy, and dashed lines denote the centers of nucleosome peaks in nuclei.</p

Methylation of genomic chromatin by PRC2 and extract.

<p>Chromatin was incubated with PRC2 or whole-cell extract, and ³H-SAM, and the product run on an SDS-PAGE gel for analysis by fluorography. PRC2 methylates its known substrate, histone H3, whereas extract methylates both histones H3 and H1.</p

Purification procedure.

<p>(A) Diagram of chromatin purification and in vitro assays. Livers were removed from rats, and used to prepare nuclei. Chromatin was then extracted by digestion with MNase and centrifugation through a sucrose gradient. (B) Flowchart of purification procedure. The first then centrifugations serve to enrich nuclei, then follows digestion with MNase to solublize the chromatin, followed by sucrose gradient-centrifugation, dialysis and concentration. Amounts indicate approximate recovery of DNA. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133246#sec011" target="_blank">Materials and Methods</a> for description of individual fractions and steps. (C) Sucrose gradient. Agarose gel of DNA from sucrose gradient fractions. (D) Total length distribution. Agarose gel of pooled fractions, run as in C. Right panel shows total distribution of fragment lengths, calculated by normalizing the signal intensity to the fragment length. Top axis shows number of nucleosomes and bottom shows length in kilobases. Dashed line represents the mean fragment length. (E) Alternative sources of chromatin. Agarose gels of sucrose fractions and electron micrographs of total chromatin from fraction S5 of material prepared from mouse ES cells and HeLa cells. Arrows show individual nucleosomes.</p

DNA sequence recovery.

<p>(A) Relative abundance of 29 genomic regions of ~100 bp, as quantified by qPCR relative to DNA in tissue. The difference in abundance between the most abundant and the least abundant sequence tested is indicated (4.3x). (B) Quantitation as in A, but using three primers within 10 kb for each of six genomic regions. The maximal difference in abundance between sites tested within 10 kb of the same chromosome is indicated (2.4x). (C) Distribution of sequences on chromosome 12, as obtained from paired-end sequencing of mononucleosomes prepared from nuclei or from purified genomic chromatin. Note that the sequences found in nuclei are also found in the purified material. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133246#pone.0133246.s001" target="_blank">S1 Fig</a>.</p

DNA methylation analysis.

<p>(A) Bisulphite sequencing analysis of genes from tissue and purified genomic chromatin. DNA was extracted (tissue) or purified as native genomic chromatin (purified) and subjected to bisulphite sequencing analysis. Yellow, blue and white boxes represent unmethylated, methylated and undetermined status of cytosine, respectively. (B) Quantification of methylation from bisulphite sequencing analysis performed in A. (C) <i>Hpa</i>II protection assay. DNA extracted from tissue or purified as native genomic chromatin was digested with methylation-sensitive restriction enzymes. Relative amount of amplified DNA from the <i>Hpa</i>II-treated sample was correlated to the relative amount of DNA amplified from the undigested sample, and expressed as percentage of protected DNA.</p

Histone mark retention.

<p>(A) Western blot of various histone marks, histone variants and core histones shows that all marks tested are detected after purification. For full lanes, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133246#pone.0133246.s002" target="_blank">S2 Fig</a>. (B) Comparative western blot shows similar levels of three histone marks and one histone variant in nuclei and in purified genomic chromatin relative to the levels of histone H3. (C) Chromatin IP of H3K4me3 and H3K27me3 in genomic chromatin and in tissue on three loci. Error bars show standard deviations from three biological replicates. Coordinate relative to TSS. (D) ChIP-Seq of H3K4me3 in genomic chromatin shows the expected pattern of peaks over active genes (arrows). (E) Metaprofile of D around the TSS shows the expected peak of H3K4me3 around the transcription start-site.</p