Nucleosomes Shape DNA Polymorphism and Divergence

<div><p>An estimated 80% of genomic DNA in eukaryotes is packaged as nucleosomes, which, together with the remaining interstitial linker regions, generate higher order chromatin structures <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004457#pgen.1004457-Lee1" target="_blank">[1]</a>. Nucleosome sequences isolated from diverse organisms exhibit ∼10 bp periodic variations in AA, TT and GC dinucleotide frequencies. These sequence elements generate intrinsically curved DNA and help establish the histone-DNA interface. We investigated an important unanswered question concerning the interplay between chromatin organization and genome evolution: do the DNA sequence preferences inherent to the highly conserved histone core exert detectable natural selection on genomic divergence and polymorphism? To address this hypothesis, we isolated nucleosomal DNA sequences from <i>Drosophila melanogaster</i> embryos and examined the underlying genomic variation within and between species. We found that divergence along the <i>D. melanogaster</i> lineage is periodic across nucleosome regions with base changes following preferred nucleotides, providing new evidence for systematic evolutionary forces in the generation and maintenance of nucleosome-associated dinucleotide periodicities. Further, Single Nucleotide Polymorphism (SNP) frequency spectra show striking periodicities across nucleosomal regions, paralleling divergence patterns. Preferred alleles occur at higher frequencies in natural populations, consistent with a central role for natural selection. These patterns are stronger for nucleosomes in introns than in intergenic regions, suggesting selection is stronger in transcribed regions where nucleosomes undergo more displacement, remodeling and functional modification. In addition, we observe a large-scale (∼180 bp) periodic enrichment of AA/TT dinucleotides associated with nucleosome occupancy, while GC dinucleotide frequency peaks in linker regions. Divergence and polymorphism data also support a role for natural selection in the generation and maintenance of these super-nucleosomal patterns. Our results demonstrate that nucleosome-associated sequence periodicities are under selective pressure, implying that structural interactions between nucleosomes and DNA sequence shape sequence evolution, particularly in introns.</p></div

Summary of the timing of CID assembly in male meiosis, showing the net gain or loss in total centromeric CID per nucleus.

<p>CID assembly occurs in prophase of meiosis I, resulting in a greater than 2-fold increase in CID at centromeres from S1 to M1 stages. CID drops by half (CID level = 2, relative to starting levels in S1) due to chromosome segregation in meiosis I; additional loss of CID is observed between telophase I and the beginning of meiosis II (CID level = 1.5). CID drops by half due to chromosome segregation in meiosis II with no assembly (CID level = 0.75). A second phase of CID assembly occurs beginning in T1 spermatids and continuing in T2–T5 spermatids in larvae (CID level = 1.25). In larvae, the level of centromeric CID in T5 haploid spermatids is comparable to the level at the S1 stage before the meiotic divisions. In adults, the level of centromeric CID in late canoe stage spermatids is also comparable to the level at the S1 stage.</p

Timing and requirements for CID assembly in mitosis and meiosis.

<p>While newly synthesized canonical histones are assembled during DNA replication in S phase, in most organisms the centromeric histone CENP-A is assembled outside of S phase, in mitosis or G1 phase. Drosophila CID is assembled at metaphase in cultured S2 cells <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001460#pbio.1001460-Mellone1" target="_blank">[13]</a>, at anaphase in the syncytial divisions in embryos <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001460#pbio.1001460-Schuh1" target="_blank">[14]</a>, and at telophase/G1 phase in larval brain nonstem and neuroblast stem cells (this study). In neuroblasts, CID loading in the mother stem cell precedes loading in the daughter cell (this study). In human mitotic HeLa cells in culture, CENP-A is assembled at late telophase/early G1 phase <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001460#pbio.1001460-Jansen1" target="_blank">[10]</a>. In meiosis in flies, CID is assembled at centromeres during prophase of meiosis I and after exit from meiosis II in spermatids (this study). The putative HJURP functional homolog in flies, CAL1, is required for CID loading in S2 cultured cells, in embryos, and in prophase of meiosis I, while HJURP is required for CENP-A loading in human cells <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001460#pbio.1001460-Foltz1" target="_blank">[16]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001460#pbio.1001460-Dunleavy1" target="_blank">[17]</a>. CENP-C is required for CID assembly in S2 cultured cells, embryos <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001460#pbio.1001460-Erhardt1" target="_blank">[24]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001460#pbio.1001460-Goshima1" target="_blank">[26]</a>, and in meiosis (this study). The timing of CENP-A assembly in somatic (stem or nonstem) cells in tissues, in embryonic or meiotic divisions in mammals is currently unknown.</p

CID assembly in male meiosis II.

<p>(A) Changes in the amount of CID at centromeres during meiosis II and in differentiating spermatids. Larval testes were fixed and stained with anti-CID antibody (green), and DNA is stained with DAPI (red). Scale bar: 1 µM. (B) Quantification of total centromeric CID fluorescence intensity per nucleus in meiosis II and in differentiating spermatids. Bars, standard errors. <i>N</i> = 799 total cells; 25, M7–M9; 114, M10–M11; 90, T1–T2; 295, T3; 196, T4–T5; 79, maturing spermatids. Note that <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001460#pbio-1001460-g002" target="_blank">Figures 2C</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001460#pbio-1001460-g004" target="_blank">4B</a> are from the same experiment and to the same scale, normalized to the initial S1 intensity value. (C) Live imaging of GFP-CID (green) and H2Av-RFP (red) expression in M10–M11 (telophase II) and differentiating spermatids (T1–T3 and T4). Scale bar: 3 µM. (D) Quantification of total centromeric GFP-CID fluorescence intensity per nucleus from live imaging of stage M10–M11 (telophase II, <i>n</i> = 24) and T1–T3 (<i>n</i> = 36) and T4 (<i>n</i> = 38) spermatids. Bars, standard errors. (E) CID localization on spermatids in stage S1 primary spermatocytes and before (early canoe stage) and after (late canoe stage) protamine exchange in adult testes fixed and stained with anti-CID antibody (green), anti-histone (blue), and DAPI (red). Scale bar: 15 µM. (F) Quantification of total centromeric CID fluorescence intensity per nucleus S1 primary spermatocytes, and early and late canoe stage spermatids in adult testes. Bars, standard errors. Values for early and late canoe are scaled to the S1 value. <i>N</i> = 144 total cells; 52, S1; 25, early canoe; 67, late canoe. (G) CID localization in mature spermatozoa. Adult testes from GFP-CID (green) transgenic flies were fixed and stained with DAPI (red). Scale bar: 5 µM.</p

Cell cycle timing of CID assembly in mitotic tissues.

<p>(A) Changes in the amount of CID at centromeres during mitosis in nonstem brain cells. Larval brains were fixed and stained with anti-CID antibody (green) and DNA is stained with DAPI (red). Scale bar: 5 µM. (B) Quantification of total centromeric CID fluorescence intensity per nucleus in stages of mitosis in dividing nonstem brain cells. Condensed chromatin at metaphase and anaphase results in a reduction in antibody penetration. Bars, standard errors. Values are normalized to the interphase average. <i>N</i> = 318 total cells. <i>N</i> = 171, interphase; 35, prophase; 52, metaphase; 21, anaphase; 39, telophase/early G1. Scale bar: 5 µM. (C) Live imaging of a nonstem brain cell from anaphase into early G1 phase expressing GFP-CID (green) and the chromatin marker H2Av-RFP (red). Time elapsed is shown in minutes. Circle indicates the initiation of CID assembly between 6 and 12 min after anaphase onset. Scale bar: 3 µM. (D) Quantification of total centromeric GFP-CID fluorescence intensity per nucleus from live imaging of dividing nonstem brain cells in larvae (<i>n</i> = 9 movies). Time elapsed in minutes after anaphase onset is shown on the <i>x</i>-axis, and fold increase in total centromeric GFP-CID intensity per nucleus is shown on the <i>y</i>-axis. Bars, standard errors.</p

Requirements of CAL1 and CENP-C for CID localization in meiosis.

<p>(A) Testes from pre-pupal larvae propagated at 29°C were fixed and stained with anti-CID (red) and anti-CENP-C (green) antibodies, and DAPI (grey). Wild type control, <i>bam-Gal4-VP-16</i>; CID-RNAi, <i>bam-Gal4-VP-16/+</i>; <i>UAS-Cid-RNAi</i>; CAL1 RNAi, <i>bam-Gal4-VP16/+</i>; <i>UAS-Cal1-RNAi</i>; CENP-C RNAi, <i>bam-Gal4-VP16/+</i>; <i>UAS-Cenp-C-RNAi</i>. Nuclei in meiotic prophase I, stages S1 and S6, are shown. White arrows indicate CENP-C localization in the nucleolus. Scale bar: 10 µM. (B) Quantitation of total centromeric CID fluorescent intensity per nucleus in wild-type, CID-, CAL1-, and CENP-C-RNAi in stages S1 and S6. For each graph, the S6 is normalized to the S1 value. <i>N</i> = control S1; 54, S6; 27, CID-RNAi S1; 92, S6; 52, CAL1-RNAi S1; 112, S6; 5, CENP-C-RNAi S1; 110, S6; 46. (C) Testes from pre-pupal larvae propagated at 29°C were fixed and stained with anti-CID (red) and anti-tubulin (green) antibodies and DAPI (grey). Representative images for chromosome segregation in meiosis I and II in control, CID-, CAL1-, and CENP-C-RNAi are shown. Scale bar: 10 µM. (D) Quantitation of the percentage of chromosome mis-segregation events in control, CID-, CAL1-, or CENP-C RNAi, in 32 cell cysts (M6 stage) after meiosis I and 64 cell cysts (T1–T3 stages) after meiosis II.</p

CAL1 and CENP-C localization in meiosis.

<p>(A) Fixed imaging of GFP-CAL1 expression/localization in prophase I in larval testes. GFP-CAL1 is localized at centromeres and in the nucleolus in stage S3 and S4 nuclei, but is reduced/delocalized from centromeres by stage S5 and almost undetectable by stage M1a (late prophase I). Colocalization of GFP-CAL1 (green) and CID (red) signals at centromeres are shown in enlarged windows. Outlines of nuclei are circled in white. Scale bar: 10 µM. (B) Fixed cell analysis of CENP-C localization in larval testes. Larval testes were fixed and stained with anti-CENP-C antibody (red), anti-CID antibody (green), and DAPI (gray). CENP-C is present at centromeres at all stages of meiosis from prophase I to telophase II (shown, stages S1, M1a, M4, M7–M9, M10–M11) but is gradually lost from centromeres beginning at T1, coinciding with the time of CID assembly (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001460#pbio-1001460-g004" target="_blank">Figure 4</a>). CENP-C is absent from centromeres in later stage T4–T5+ spermatids (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001460#pbio.1001460.s004" target="_blank">Figure S4</a>). Scale bar: 5 µM, S1–M4; 1 µm, M7–M9–T5+.</p

Chromosome 4 has a very low incidence of polymerase pausing identified by GRO-seq data.

<p>A. The bar graph shows the percentage of transcripts associated with RNA polymerase (grey) and the percentage of those RNA polymerase-associated genes exhibiting significant pausing (black) for euchromatin, pericentric heterochromatin, and chromosome 4. A pausing index (PI) threshold value of 10 was used. B. The low frequency of polymerase pausing observed for chromosome 4 is independent of the PI selected. C. The average T_m of 9-mers downstream of the TSS for chromosome 4 genes tends to be lower than that of other genes. T_m (Y-axis) of 9-mers in the 100 bp downstream of the TSS (bp, X-axis) is compared for genes on chromosome 4 (red) to genes classified as either paused (black) or not-paused (grey) by GRO-seq analysis. Data from S2 cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002954#pgen.1002954-Roy1" target="_blank">[23]</a>.</p

Lack of HP1a does not lead to a loss of POF from chromosome 4.

<p>A. H3K9me2 and H3K9me3 levels decrease in HP1a mutants, while POF enrichment is not reduced. The smoothed M-value (Y-axis) is shown for pericentric heterochromatin (right) and chromosome 4 (left) comparing wildtype (dark color) and trans-heterozygous <i>Su(var)205⁰⁴/Su(var)205⁰⁵</i> mutants. Error bars: SEM. B. Browser shot illustrating the retention of POF enrichment on chromosome 4 in HP1a mutants (top panel) and depletion of H3K9me2 and H3K9me3 both in chromosome 4 (top) and pericentric heterochromatin (bottom). The M-value scale (Y-axis) is identical for wildtype and mutant ranging from 0 to 3. C. Changes in H3K9me2, H3K9me3, and HP1a enrichment at TSSs of actively transcribed genes, over gene bodies of active genes, and in silent regions on chromosome 4 in HP1a mutant. Error bars: SEM.</p

Lack of EGG leads to large-scale changes in POF, HP1a, and H3K9 methylation specifically on chromosome 4.

<p>A. Depletion of EGG alters H3K9me2/3, HP1a, and POF enrichment. Scaled enrichment is shown on chromosome 4 (left) and in pericentric heterochromatin (right) comparing wildtype (dark color) and <i>egg 10.1-1a</i> homozygous mutant (light color). Error bars: SEM. B. Browser shots showing the reduction of POF, HP1a, and H3K9me2/3 on chromosome 4 (top panel) and the relatively small change in pericentric heterochromatin observed in <i>egg</i> mutants. The M-value scale (Y-axis) is identical for wildtype and mutant ranging from 0 to 3. C. The 70 kb proximal region on chromosome 4 shows minimal changes in POF, HP1a, and H3K9me2/3 levels in <i>egg</i> mutants, distinct from the alterations in the remainder of the chromosome illustrated in B. The M-value scale (Y-axis) is identical for wildtype and mutant ranging from 0 to 3. D. Changes in H3K9me2/me3, HP1a, and POF enrichment (Y-axis: smoothed M-values) are examined separately for TSS of actively transcribed genes, their gene bodies, and silent regions on chromosome 4. Error bars: SEM. Data from third instar larvae.</p