Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these \u27proto-chromatosomes\u27 are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1

The enrichment of the reference loci and the EUS regions of the <i>CYP19</i> locus in individual SEVENS fractions.

<p>(A-C) The fractional distribution of the active reference loci (panels in the first row), the repressed reference loci (panels in the second row), and the EUS regions (panels in the third to fifth rows) in HepG2 (A), KGN (B), or HeLa cells (C) was represented as the fold enrichment in each fraction relative to an average calculated from total fractions by using a log2 scale.</p

The Proportion of Chromatin Graded between Closed and Open States Determines the Level of Transcripts Derived from Distinct Promoters in the <i>CYP19</i> Gene

<div><p>The human <i>CYP19</i> gene encodes aromatase, which converts androgens to estrogens. <i>CYP19</i> mRNA variants are transcribed mainly from three promoters. Quantitative RT-PCR was used to measure the relative amounts of each of the three transcripts and determine the on/off state of the promoters. While some of the promoters were silent, <i>CYP19</i> mRNA production differed among the other promoters, whose estimated transcription levels were 0.001% to 0.1% of that of the <i>TUBB</i> control gene. To investigate the structural aspects of chromatin that were responsible for this wide range of activity of the <i>CYP19</i> promoters, we used a fractionation protocol, designated SEVENS, which sequentially separates densely packed nucleosomes from dispersed nucleosomes. The fractional distribution of each inactive promoter showed a similar pattern to that of the repressed reference loci; the inactive regions were distributed toward lower fractions, in which closed chromatin comprising packed nucleosomes was enriched. In contrast, active <i>CYP19</i> promoters were raised toward upper fractions, including dispersed nucleosomes in open chromatin. Importantly, these active promoters were moderately enriched in the upper fractions as compared to active reference loci, such as the <i>TUBB</i> promoter; the proportion of open chromatin appeared to be positively correlated to the promoter strength. These results, together with ectopic transcription accompanied by an increase in the proportion of open chromatin in cells treated with an H3K27me inhibitor, indicate that <i>CYP19</i> mRNA could be transcribed from a promoter in which chromatin is shifted toward an open state in the equilibrium between closed and open chromatin.</p></div

The principle of the SEVENS assay.

<p>(A) Neighboring nucleosomes are crosslinked to each other by formaldehyde. Because this crosslinking reagent has a short arm of a single carbon, it preferentially reacts to condensed nucleosomes in closed chromatin. Thus, following solubilization with a detergent and mild sonication, condensed nucleosomes that are sufficiently crosslinked form a large particle, while nucleosomes in open chromatin escape from the crosslinking reaction to be a small particle. When such a preparation is subjected to sedimentation velocity centrifugation with a sucrose density gradient, chromatin is fractionated with regard to the size of sheared particles, namely to the crowdedness of nucleosomes (closed chromatin vs. open chromatin). (B and C) Chromatin structure comprises nucleosomes in equilibrium between a condensed and a dispersed state. Because a preparation for the assay contains multiple fragments of crosslinked chromatin obtained from more than a million cells, data analyzed for a locus-of-interest are represented as the proportion of chromatin graded between open and closed chromatin. Thus, the fractional distribution of a locus in closed chromatin gradually increases toward lower fractions (B). Conversely, a locus in open chromatin increases toward upper fractions (C).</p

Transcription from each of three <i>CYP19</i> promoters.

<p>(A) The gene structure of the human <i>CYP19</i> locus. Closed boxes with Roman numerals represent exons of the gene. Primers for conventional RT-PCR are denoted as arrows; the three rightward arrows are forward primers for the respective first exons, while the leftward arrow is a reverse primer for the common second exon. (B) Conventional RT-PCR analyses revealed the existence of three kinds of <i>CYP19</i> transcripts. “Ia-II”, “Ib-II”, or “Ic-II” indicates a PCR reaction amplified with a primer set annealing to exons Ia/II, exons Ib/II, or exons Ic/II, respectively. PCR for the “<i>TUBB</i>” (β-tubulin) gene was performed as a control. “M” denotes a molecular weight marker. (C) Quantitative RT-PCR analyses revealed a comparable level of the <i>TUBB</i> transcripts among HepG2, KGN, and JEG-3 cell lines following normalization to the amount of 18S rRNA. The quantitative similarity was confirmed after performing Student’s t-test (p > 0.05). The value for the transcripts in the three cell lines is presented as a percentage of that in HeLa cells. (D) The relative amount of each kind of the <i>CYP19</i> transcript was estimated by using quantitative RT-PCR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128282#sec002" target="_blank">Materials and Methods</a>). “Ia”, “Ib”, or “Ic” indicates a PCR reaction for transcripts initiated from the exon Ia, Ib, or Ic, respectively. The values in the chart denote a percentage of the amount of the <i>TUBB</i> transcripts in each cell line. “ND” means “not detected”. (E) The amount of aromatase, which is the product of the <i>CYP19</i> gene, was estimated using western blotting analyses. As a loading control, blotting with anti-β-actin was also performed. (F) By using flow cytometry analyses, the homogeneity of the expression of the <i>CYP19</i> gene in each cell line was analyzed. Only in JEG-3 cells, a cell population extra-highly expressing the <i>CYP19</i> gene was detected (arrow in the panel of JEG-3). The open or the filled gray histogram denotes cell populations stained with mouse IgG isotype control or anti-aromatase antibody, respectively.</p

Chromatin structure observed in the SEVENS assay.

<p>(A-C) The SEVENS assays revealed distinct chromatin structures in the <i>CYP19</i> locus of HepG2 (A), KGN (B), and HeLa cells (C). The proportion of open and closed chromatin is represented as an enrichment ratio of a given sequence in upper fractions to that in lower fractions (Up/Lo) using a log2 scale (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128282#sec002" target="_blank">Materials and Methods</a>). The gene structure of the <i>CYP19</i> locus is drawn above each chart. Each vertical line extends from a promoter region to note a corresponding position in the respective lower charts. Red circles and blue rectangles denote the Up/Lo values of active and repressed reference loci, respectively, which are abbreviated as TU, AC, GA, OR, MY, or IL for the <i>TUBB</i>, <i>ACTB</i>, <i>GAPDH</i>, <i>OR1A1</i>, <i>MYT1</i>, or <i>IL2RA</i> loci, respectively. To compare assessed regions of the <i>CYP19</i> locus to these references, red and blue belts are placed on the charts to indicate the ranges of the Up/Lo values of the references. The positions referred to in the text are labeled as “<u>e</u>nrichment in <u>u</u>pper fractions of the <u>S</u>EVENS assay (EUS)” in the charts.</p

The nucleosome occupancy in the <i>CYP19</i> locus.

<p>(A-C) ChIP assays with anti-pan-histone H3 revealed the amount of nucleosomes in the <i>CYP19</i> locus in HepG2 (A), KGN (B), or HeLa cells (C). The nucleosome level is expressed as a percentage of total input chromatin (IP/Input). The solid line in the respective charts represents the average value of IP/Input. The level at the <i>OR1A1</i> (■) or the <i>TUBB</i> (●) locus as a reference is also plotted at the right of each chart. To compare a given region in the <i>CYP19</i> locus to the references, broken lines are drawn from the reference marks. The gene structure of <i>CYP19</i> and the EUS regions are also represented on each chart.</p

The distribution of H3K27me3 and H3K27ac in the <i>CYP19</i> locus.

<p>(A-C) ChIP assays with anti-H3K27me3 (blue lines) or anti-H3K27ac (red lines) revealed the distribution of these histone modifications in the <i>CYP19</i> locus in HepG2 (A), KGN (B), or HeLa cells (C). Following normalization to the amount of total histone H3, relative occupancy of H3K27me3 or H3K27ac is represented by using a percentage of the modification at the <i>MYT1</i> or the <i>TUBB</i> control locus, respectively. Note that the direction of the y-axis of the charts for H3K27me3 is downward. The gene structures of <i>CYP19</i> and the EUS regions are also represented on each chart.</p

Complex Cell Cycle Abnormalities Caused by Human T-Lymphotropic Virus Type 1 Tax▿ §

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL), a malignancy of CD4+ T cells whose etiology is thought to be associated with the viral trans-activator Tax. We have shown recently that Tax can drastically upregulate the expression of p27Kip1 and p21CIP1/WAF1 through protein stabilization and mRNA trans-activation and stabilization, respectively. The Tax-induced surge in p21CIP1/WAF1 and p27Kip1 begins in S phase and results in cellular senescence. Importantly, HeLa and SupT1 T cells infected by HTLV-1 also arrest in senescence, thus challenging the notion that HTLV-1 infection causes cell proliferation. Here we use time-lapse microscopy to investigate the effect of Tax on cell cycle progression in two reporter cell lines, HeLa/18x21-EGFP and HeLa-FUCCI, that express enhanced green fluorescent protein (EGFP) under the control of 18 copies of the Tax-responsive 21-bp repeat element and fluorescent ubiquitin cell cycle indicators, respectively. Tax-expressing HeLa cells exhibit elongated or stalled cell cycle phases. Many of them bypass mitosis and become single senescent cells as evidenced by the expression of senescence-associated β-galactosidase. Such cells have twice the normal equivalent of cellular contents and hence are enlarged, with exaggerated nuclei. Interestingly, nocodazole treatment revealed a small variant population of HeLa/18x21-EGFP cells that could progress into mitosis normally with high levels of Tax expression, suggesting that genetic or epigenetic changes that prevent Tax-induced senescence can occur spontaneously at a detectable frequency