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Self-assembly of thin plates from micrococcal nuclease-digested chromatin of metaphase chromosomes.

ABSTRACT: The three-dimensional organization of the enormously long DNA molecules packaged within metaphase chromosomes has been one of the most elusive problems in structural biology. Chromosomal DNA is associated with histones and different structural models consider that the resulting long chromatin fibers are folded forming loops or more irregular three-dimensional networks. Here, we report that fragments of chromatin fibers obtained from human metaphase chromosomes digested with micrococcal nuclease associate spontaneously forming multilaminar platelike structures. These self-assembled structures are identical to the thin plates found previously in partially denatured chromosomes. Under metaphase ionic conditions, the fragments that are initially folded forming the typical 30-nm chromatin fibers are untwisted and incorporated into growing plates. Large plates can be self-assembled from very short chromatin fragments, indicating that metaphase chromatin has a high tendency to generate plates even when there are many discontinuities in the DNA chain. Self-assembly at 37°C favors the formation of thick plates having many layers. All these results demonstrate conclusively that metaphase chromatin has the intrinsic capacity to self-organize as a multilayered planar structure. A chromosome structure consistent of many stacked layers of planar chromatin avoids random entanglement of DNA, and gives compactness and a high physical consistency to chromatids.

SUBMITTER: Milla M

PROVIDER: S-EPMC3414884 | biostudies-other | 2012 Aug

REPOSITORIES: biostudies-other

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Self-assembly of thin plates from micrococcal nuclease-digested chromatin of metaphase chromosomes.

Milla Maria M Daban Joan-Ramon JR

Biophysical journal 20120801 3

The three-dimensional organization of the enormously long DNA molecules packaged within metaphase chromosomes has been one of the most elusive problems in structural biology. Chromosomal DNA is associated with histones and different structural models consider that the resulting long chromatin fibers are folded forming loops or more irregular three-dimensional networks. Here, we report that fragments of chromatin fibers obtained from human metaphase chromosomes digested with micrococcal nuclease ...[more]

PMID: 22947873

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Project description:BackgroundChromatin-modifying reagents that alter histone associating proteins, DNA conformation or its sequence are well established strategies for studying chromatin structure in interphase (G1, S, G2). Little is known about how these compounds act during metaphase. We assessed the effects of these reagents at genomic loci that show reproducible, non-random differences in accessibility to chromatin that distinguish homologous targets by single copy DNA probe fluorescence in situ hybridization (scFISH). By super-resolution 3-D structured illumination microscopy (3D-SIM) and other criteria, the differences correspond to 'differential accessibility' (DA) to these chromosomal regions. At these chromosomal loci, DA of the same homologous chromosome is stable and epigenetic hallmarks of less accessible interphase chromatin are present.ResultsTo understand the basis for DA, we investigate the impact of epigenetic modifiers on these allelic differences in chromatin accessibility between metaphase homologs in lymphoblastoid cell lines. Allelic differences in metaphase chromosome accessibility represent a stable chromatin mark on mitotic metaphase chromosomes. Inhibition of the topoisomerase IIα-DNA cleavage complex reversed DA. Inter-homolog probe fluorescence intensity ratios between chromosomes treated with ICRF-193 were significantly lower than untreated controls. 3D-SIM demonstrated that differences in hybridized probe volume and depth between allelic targets were equalized by this treatment. By contrast, DA was impervious to chromosome decondensation treatments targeting histone modifying enzymes, cytosine methylation, as well as in cells with regulatory defects in chromatid cohesion. These data altogether suggest that DA is a reflection of allelic differences in metaphase chromosome compaction, dictated by the localized catenation state of the chromosome, rather than by other epigenetic marks.ConclusionsInhibition of the topoisomerase IIα-DNA cleavage complex mitigated DA by decreasing DNA superhelicity and axial metaphase chromosome condensation. This has potential implications for the mechanism of preservation of cellular phenotypes that enables the same chromatin structure to be correctly reestablished in progeny cells of the same tissue or individual.

Project description:BackgroundCondensation differences along the lengths of homologous, mitotic metaphase chromosomes are well known. This study reports molecular cytogenetic data showing quantifiable localized differences in condensation between homologs that are related to differences in accessibility (DA) of associated DNA probe targets. Reproducible DA was observed for ~10% of locus-specific, short (1.5-5 kb) single copy DNA probes used in fluorescence in situ hybridization.ResultsFourteen probes (from chromosomes 1, 5, 9, 11, 15, 17, 22) targeting genic and intergenic regions were developed and hybridized to cells from 10 individuals with cytogenetically-distinguishable homologs. Differences in hybridization between homologs were non-random for 8 genomic regions (RGS7, CACNA1B, GABRA5, SNRPN, HERC2, PMP22:IVS3, ADORA2B:IVS1, ACR) and were not unique to known imprinted domains or specific chromosomes. DNA probes within CCNB1, C9orf66, ADORA2B:Promoter-Ex1, PMP22:IVS4-Ex 5, and intergenic region 1p36.3 showed no DA (equivalent accessibility), while OPCML showed unbiased DA. To pinpoint probe locations, we performed 3D-structured illumination microscopy (3D-SIM). This showed that genomic regions with DA had 3.3-fold greater volumetric, integrated probe intensities and broad distributions of probe depths along axial and lateral axes of the 2 homologs, compared to a low copy probe target (NOMO1) with equivalent accessibility. Genomic regions with equivalent accessibility were also enriched for epigenetic marks of open interphase chromatin (DNase I HS, H3K27Ac, H3K4me1) to a greater extent than regions with DA.ConclusionsThis study provides evidence that DA is non-random and reproducible; it is locus specific, but not unique to known imprinted regions or specific chromosomes. Non-random DA was also shown to be heritable within a 2 generation family. DNA probe volume and depth measurements of hybridized metaphase chromosomes further show locus-specific chromatin accessibility differences by super-resolution 3D-SIM. Based on these data and the analysis of interphase epigenetic marks of genomic intervals with DA, we conclude that there are localized differences in compaction of homologs during mitotic metaphase and that these differences may arise during or preceding metaphase chromosome compaction. Our results suggest new directions for locus-specific structural analysis of metaphase chromosomes, motivated by the potential relationship of these findings to underlying epigenetic changes established during interphase.

Dataset Information

Self-assembly of thin plates from micrococcal nuclease-digested chromatin of metaphase chromosomes.

Publications

Self-assembly of thin plates from micrococcal nuclease-digested chromatin of metaphase chromosomes.

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