Comparative Analysis of Genome-Wide Chromosomal Histone Modification Patterns in Maize Cultivars and Their Wild Relatives

<div><p>Recent advances demonstrate that epigenome changes can also cause phenotypic diversity and can be heritable across generations, indicating that they may play an important role in evolutionary processes. In this study, we analyzed the chromosomal distribution of several histone modifications in five elite maize cultivars (B73, Mo17, Chang7-2, Zheng58, ZD958) and their two wild relatives (<i>Zea mays</i> L. ssp. <i>parviglumis</i> and <i>Zea nicaraguensis</i>) using a three-dimensional (3D) epigenome karyotyping approach by combining immunostaining and 3D reconstruction with deconvolution techniques. The distribution of these histone modifications along chromosomes demonstrated that the histone modification patterns are conserved at the chromosomal level and have not changed significantly following domestication. The comparison of histone modification patterns between metaphase chromosomes and interphase nuclei showed that some of the histone modifications were retained as the cell progressed from interphase into metaphase, although remodelling existed. This study will increase comprehension of the function of epigenetic modifications in the structure and evolution of the maize genome.</p></div

Comparison of H3K4me3 distribution of across interphase nuclei and metaphase chromosomes in B73.

<p>Immunolabelled metaphase chromosomes (DAPI staining signals in blue and immunosignals in green) by H3K4me3 are aligned with the distribution of H3K4me3 and DNA methylation in the equivalent interphase nuclei assembled from ChIP-seq data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097364#pone.0097364-Wang1" target="_blank">[31]</a>. Each chromosome was divided into 10 Mb intervals. The left vertical axis indicates the number of unique reads per 10 Mb of H3K4me3 (blue curve), and the right vertical axis indicates the number of unique reads per 10 Mb of DNA methylation (red curve).</p

TSA-treated plants are more sensitive to freezing.

<p>A. Plants were freeze-treated at the indicated temperatures for 15 minutes and then the survival of plants was quantified. B. Leakage of electrolytes from normal and TSA-treated plants was estimated by determining relative electro-conductivity of the bathing solution of the leaf tissue. Bars represent standard errors.</p

Ideogram of FISH karyotype of four maize lines.

<p>The four maize lines include B73, Mo17, Zheng58 and ZD958 (Chang7-2×Zheng58). Assignments of pseudo-colors to each probe: TAG as white, CentC as green, 45S rDNA as yellow and knob 180-bp as red.</p

Primers used for quantitative real-time PCR, CHART-PCR and ChIP.

<p>Primers used for quantitative real-time PCR, CHART-PCR and ChIP.</p

Comparison of H3K9Ac, H4K5Ac and H44Ac levels in the <i>ZmICE1</i> and <i>ZmCOR413</i> gene regions.

<p>Cold stress caused hyperacetylation in the <i>ZmICE1</i> and <i>ZmCOR413</i> promoter regions and the first exon. Additionally, these two regions were still hyperacetylated after cold-acclimated plants were treated with TSA. No significant changes in histone acetylation were detected in TSA-treated normal plants. Bars represent standard errors.</p

HDACs selectively regulate <i>ZmDREB1</i> gene expression.

<p>A–C. Transcript levels of stress responsive genes after different treatments were measured by real-time PCR. Compared with the cold-responsive genes <i>ZmDREB1</i> and <i>ZmCOR413</i>, the expression of <i>ZmICE1</i> was not significantly affected by TSA treatment. D–E. <i>ZmDBF1</i> and <i>rab17</i> were highly induced by ABA, NaCl and mannitol treatments, but not by cold stress. These genes did not demonstrate significant changes in expression after TSA treatment. F. The time course profile of <i>ZmDREB1</i> and <i>ZmCOR413</i> expression under TSA treatment. TSA treatment rapidly represses <i>ZmDREB1</i> and slowly represses <i>ZmCOR413</i>. Samples were taken from 7-day-old seedlings grown under 25°C (x-axis 0), and from plants treated with different reagents or stresses at the indicated time points. The data are the mean ± standard error for triplicate quantitative PCR reactions for each time point from three independent experiments.</p

Western blot assay demonstrates the change in histone acetylation levels during cold acclimation.

<p>A. Protein samples were extracted from 7-day-old seedlings grown under 25°C (0 day), from seedlings that were further cold-treated at for 1–4 days, and from seedlings returned to 25°C for 1–4 days after cold treatment. Global acetylation levels of histones H3 and H4 were decreased after cold treatment and were recovered when seedlings were returned to 25°C. B. Reference cultivation without cold treatment was performed in parallel, showing that the histone acetylation levels underwent little change. Beta actin and total histone H3 were used as equal loading controls, and representative blots are shown. C. Deacetylation of histones H3 and H4 is revealed by immunostaining representative interphase nuclei with specific antibodies. DAPI was used as a counterstain. The ‘DAPI’ panel shows DAPI-stained DNA images, the ‘H3K9Ac’, ‘H44Ac’ and ‘H4K5Ac’ panels show immunostained images, and the ‘Merge’ panel shows a combination of blue and green signals. Under normal growth conditions, the strongly acetylated histones H3 and H4 signals were evenly distributed in the nucleus, and nucleoli were barely acetylated. When deacetylation of histones H3 and H4 occurred, weak acetylation signals were observed during cold acclimation. The histone acetylation levels were recovered after the seedlings were returned to 25°C (Bar = 10 µm). D. Histogram showing the mean gray values of the immunostaining signals for histone acetylation. Error bars represent the standard error of the mean. More than 500 nuclei were analyzed.</p

Comparison of H3K4me3 and FISH signal distribution in <i>Z. mays</i> ssp. <i>parviglumis</i> and <i>Z. nicaraguensis</i>.

<p>(A, E) 2D image of DAPI staining (blue) signals and H3K4me3 signals (green). (B, F) Image of DAPI staining (blue) signals and H3K4me3 signals (green) after 3D deconvolution. (C, G) Metaphase chromosome identification by combination of the four probes and DAPI staining; Assignments of pseudo-colors to each probe: TAG as white (pseudo-color), CentC as green, 45S rDNA as yellow (pseudo-color) and knob 180-bp as red. (D, H) H3K4me3 and FISH karyotypes in <i>Z. mays</i> ssp. <i>parviglumis</i> and <i>Z. nicaraguensis</i>. Scale bar = 10 µm.</p

Comparison of the chromosomal distribution of six histone modifications of five maize lines.

<p>The 3D immunolabelled (H3K4me2, H3K4me3, H3K9ac, H4K5ac, H3K9me2, and H3K27me2) and DAPI stained chromosomes of four maize inbred lines (B73, Mo17, Chang7-2 and Zheng58) and a hybrid: ZD958 (Chang7-2×Zheng58)) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097364#pone.0097364.s001" target="_blank">figure S1</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097364#pone.0097364.s006" target="_blank">figure S6</a> were arranged in the order of the maize chromosome number (1−10). Immunostaining signals in green and DAPI staining signals in blue. Scale bar = 10 µm.</p