Histone Modifications within the Human X Centromere Region

Human centromeres are multi-megabase regions of highly ordered arrays of alpha satellite DNA that are separated from chromosome arms by unordered alpha satellite monomers and other repetitive elements. Complexities in assembling such large repetitive regions have limited detailed studies of centromeric chromatin organization. However, a genomic map of the human X centromere has provided new opportunities to explore genomic architecture of a complex locus. We used ChIP to examine the distribution of modified histones within centromere regions of multiple X chromosomes. Methylation of H3 at lysine 4 coincided with DXZ1 higher order alpha satellite, the site of CENP-A localization. Heterochromatic histone modifications were distributed across the 400–500 kb pericentromeric regions. The large arrays of alpha satellite and gamma satellite DNA were enriched for both euchromatic and heterochromatic modifications, implying that some pericentromeric repeats have multiple chromatin characteristics. Partial truncation of the X centromere resulted in reduction in the size of the CENP-A/Cenp-A domain and increased heterochromatic modifications in the flanking pericentromere. Although the deletion removed ∼1/3 of centromeric DNA, the ratio of CENP-A to alpha satellite array size was maintained in the same proportion, suggesting that a limited, but defined linear region of the centromeric DNA is necessary for kinetochore assembly. Our results indicate that the human X centromere contains multiple types of chromatin, is organized similarly to smaller eukaryotic centromeres, and responds to structural changes by expanding or contracting domains

H4K20 trimethylation (H4K20me3) at the X centromere in two human cell lines and one of the mouse-human somatic cell hybrids.

<p>Each number across the schematic representation of the centromere is a genomic site that was interrogated by ChIP-PCR. Control regions, including GAPDH, AFM and X-linked ZXDA are also included. The bar graph shows relative enrichment for H3K27 methylation (n = 3 with SD) calculated as percentage of input.</p

Cell lines included in the study.

<p>Cell lines included in the study.</p

H3K27 trimethylation (H3K27me3) at the X centromere in human cells and mouse-human somatic cell hybrids.

<p>Each number across the schematic representation of the centromere is a genomic site that was interrogated by ChIP-PCR. The bar graph shows relative enrichment for H3K27 methylation (n = 3 with SD). Control regions, including GAPDH, AFM and X-linked ZXDA are also included. The bar graph shows relative enrichment for H3K27 methylation (n = 3 with SD) calculated as percentage of input.</p

H3K9me2 enrichment at X centromeres in human cell lines and mouse-human hybrids.

<p>The schematic shows the structure of the X centromere. Each number along the centromere represents a genomic site or control region that was interrogated by ChIP-PCR with a specific histone antibody. Each bar graph shows relative enrichment for each histone modification (n≥3 with SD). Control regions, including GAPDH, AFM and X-linked ZXDA are also included. The bar graph shows enrichment calculated as percentage of input.</p

Comparision of histone modifications across a normal and deleted version of the same X centromere.

<p>The schematics at the top show the structure of the normal <i>(A)</i> and partially deleted <i>(B)</i> X centromere. Each chromosome is contained in a human-hamster cell line. The numbers along the centromere represents genomic sites interrogated by ChIP-PCR with a specific histone antibody. The line plots shows average relative enrichment for each histone modification across the centromere region (n = 3 with SD). Control regions, including GAPDH and and X-linked ZXDA are also included. The bar graph shows relative enrichment for H3K27 methylation (n = 3 with SD) calculated as percentage of input. Asterisks indicate statistically significant differences in enrichment for a modification at a particular site between the normal X and deleted X as determined by a Student's t-test (<i>p</i><0.05).</p

H3K27 mono-methylation (H3K27me1) at the X centromere in human cells and mouse-human somatic cell hybrids.

<p>Each number across the schematic representation of the centromere is a genomic site that was interrogated by ChIP-PCR. Control regions, including GAPDH, AFM and X-linked ZXDA are also included. The bar graph shows relative enrichment for H3K27 methylation (n = 3 with SD) calculated as percentage of input.</p

CENP-A mapping on X alpha satellite (DXZ1) on normal and partially deleted X chromosomes in a human-hamster somatic cell hybrid.

<p><i>(A)</i> Schematic of the genomic structure of the X centromeres s of the normal X chromosome in the human:hamster cell line HTM18TC8 and of its 2.7 Mb minichromosome derivative retained in the hamster somatic cell hybrid FA3Wg8-4. <i>(B)</i> IF-FISH on two representative chromatin fibers from the normal X chromosome showing that hamster Cenp-A (red) was localized to a portion of DXZ1 (green), and asymmetrically distributed. Each fiber shown represents an independent experiment. Arrowed lines denote Cenp-A staining (red). Scale bar is 10 microns. <i>(C)</i> IF-FISH on two representative chromatin fibers from the truncated X chromosome showed that CENP-A remained asymmetrically distributed on DXZ1. Each fiber shown represents an independent experiment. Scale bar is 5 microns. <i>(D)</i> Genomic sizes of CENP-A binding domains (gray) and DXZ1 arrays (black) on the normal X before and after partial centromere deletion. N = 15 fibers for normal X and n = 20 fibers for deleted X. The asterisk indicates that reduction in CENP-A array size on the deleted X is statisically significant as determined by a Student's t-test (<i>p</i> = 0.01).</p

Primer sets used for ChIP-PCR.

<p>Primer sets used for ChIP-PCR.</p