Nick Gilbert ^{1, 3}, Shelagh Boyle ^{1, 3}, Heike Fiegler ², Kathryn Woodfine ², Nigel P. Carter ², and Wendy A.
Bickmore ^{1, *}

¹ MRC Human Genetics Unit, Edinburgh, EH4 2XU, Scotland
² The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom
³ These authors contributed equally to this work.

^*Correspondence:  w.bickmore@hgu.mrc.ac.uk

We present an analysis of chromatin fiber structure across the human genome. Compact and open chromatin fiber structures were separated by sucrose sedimentation and their distributions analyzed by hybridization to metaphase chromosomes and genomic microarrays. We show that compact chromatin fibers originate from some sites of heterochromatin (C-bands), and G-bands (euchromatin). Open chromatin fibers correlate with regions of highest gene density, but not with gene expression since inactive genes can be in domains of open chromatin, and active genes in regions of low gene density can be embedded in compact chromatin fibers. Moreover, we show that chromatin fiber structure impacts on further levels of chromatin condensation. Regions of open chromatin fibers are cytologically decondensed and have a distinctive nuclear organization. We suggest that domains of open chromatin may create an environment that facilitates transcriptional activation and could provide an evolutionary constraint to maintain clusters of genes together along chromosomes.

Figure 1. Sucrose Gradient Fractionation of Human Chromatin

(A) MNase digestion of nuclei was
         used to produce chromatin fragments with a size of ~20 kb. The soluble chromatin from the digest
         marked by an asterisk, was run on a 6–40% isokinetic sucrose gradient. For two chromatin
         fragments of equal length (kb) the more open/disordered fragment (top) will sediment slower than
         the more compact/rigid one (bottom)

(B) The gradient was fractionated from top to bottom and the
         DNA purified from each fraction examined by agarose gel electrophoresis.

(C) To isolate DNA
         fragments from the same fraction (sedimentation rate), but with different lengths (and thus
         different chromatin fiber conformations), DNA from a gradient fraction (asterisked in B) was size
         selected by PFGE. DNA was purified from a gel slice corresponding to the peak of EtBr staining
         (bulk chromatin). To represent "compact" chromatin, DNA was purified from a gel slice
         containing fragments ~10 kb shorter than the EtBr peak. DNA from "open" chromatin was purified
         from a gel slice containing fragments ~10 kb longer than bulk chromatin.
...

FISH with Total DNA and Input Chromatin Probes

Supplemental Figure S1. FISH with Total DNA and Input Chromatin Probes

Hybridization of metaphase chromosome spreads, in the absence (-Cot1) or presence (+Cot1) of 50 mg
human Cot1, with biotin-dUTP labeled probes prepared from total human genomic DNA (A) or from input chromatin labelled with biotin-dUTP (B) or biotin-dCTP (C). Chromosomes were identified from the reverse DAPI banding (middle panels). Uniform labeling of chromosome arms is seen with each probe in the presence of suppression by Cot1, but dCTP-labeled probe fails to hybridize strongly to constitutive heterochromatin, even in the absence of Cot1 suppression.

The log₂ ratio for open: input chromatin after hybridization to a whole human genome microarray assembled from clones at 1 Mb intervals (Fielgler et al., 2003; Woodfine et al., 2004). The data for chromosome 1 from two independent experiments, each with color reversal is shown.

To exclude erroneous points from analysis, the correlation between individual spots on the microarray for
colour reversal hybridizations was determined for two independent experiments. For each correlation the
color-reversed data sets were normalized to each other and points falling out with 2 S.D. were excluded
from further analysis. 

Supplemental Figure S4. Distribution of Open Chromatin across the Whole Human Genome by Hybridization to a Whole Genome Microarray

The log₂ ratio for open:input chromatin, averaged from two experiments each with color reversal, after hybridization to a whole human genome microarray assembled from clones at 1 Mb intervals (Fielgler et al.,
2003; Woodfine et al., 2004). The dotted line indicates a 1:1 ratio of open:input hybridization signal
(log₂=0). Ideograms of each chromosome are aligned to each graph with T bands highlighted in red.
G bands are black and C-bands yellow.

To exclude erroneous points from analysis, the correlation between individual spots on the microarray for
color reversal hybridizations was determined. For each correlation, the color-reversed data sets were
normalized to each other and points falling out with 2 S.D. were excluded from further analysis.

The ratio of open:input chromatin was calculated from the average of two independent fractionations of open chromatin, each hybridised twice with colour reversal to the 1Mb microarray. These values were averaged across each chromosome. Genomic DNA (female):input chromatin (male) hybridisation ratios per chromosome demonstrate that there is not preferential release of chromatin from gene-rich chromosomes, since ratios for autosomes approximate to 1, compared with the sex chromosomes (*). Gene density per chromosome was calculated from ENSEMBL, and the GC content of each chromosome was taken from Venter et al. (2001). The mean replication time for each chromosome was determined using the same whole  genome microarray (Woodfine et al., 2004). Gene expression data for lymphoblasts (Woodfine et al., 2004) was expressed both as average expression level (arbitrary units) or probability of expression for all genes assayed per chromosome.

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"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".

Links to RNA and Biological Causality:

Further Topics in:  Euchromatin,  active DNA, and  RNA  ribo-regulators:

Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".

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