Gene density and transcription influence the localization of chromatin outside of chromosome territories detectable by FISH.

Abstract

Genes can be transcribed from within chromosome territories; however, the major histocompatibilty complex locus has been reported extending away from chromosome territories, and the incidence of this correlates with transcription from the region. A similar result has been seen for the epidermal differentiation complex region of chromosome 1. These data suggested that chromatin decondensation away from the surface of chromosome territories may result from, and/or may facilitate, transcription of densely packed genes subject to coordinate regulation.To investigate whether localization outside of the visible confines of chromosome territories can also occur for regions that are not coordinately regulated, we have examined the spatial organization of human 11p15.5 and the syntenic region on mouse chromosome 7. This region is gene rich but its genes are not coordinately expressed, rather overall high levels of transcription occur in several cell types. We found that chromatin from 11p15.5 frequently extends away from the chromosome 11 territory. Localization outside of territories was also detected for other regions of high gene density and high levels of transcription. This is shown to be partly dependent on ongoing transcription. We suggest that local gene density and transcription, rather than the activity of individual genes, influences the organization of chromosomes in the nucleus.

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The Journal of Cell Biology, Volume 159, Number 5, December 9, 2002 753–763
http://www.jcb.org/cgi/doi/10.1083/jcb.200207115
The Rockefeller University Press, 0021-9525/2002/12/753/11 $5.00
JCB
Article
753
Gene density and transcription influence the
localization of chromatin outside of chromosome
territories detectable by FISH
Nicola L. Mahy, Paul E. Perry, and Wendy A. Bickmore
MRC Human Genetics Unit, Edinburgh EH4 2XU, UK
enes can be transcribed from within chromosome
territories; however, the major histocompatibilty
complex locus has been reported extending away
from chromosome territories, and the incidence of this
correlates with transcription from the region. A similar result
has been seen for the epidermal differentiation complex
region of chromosome 1. These data suggested that chromatin
decondensation away from the surface of chromosome
territories may result from, and/or may facilitate, transcription
of densely packed genes subject to coordinate regulation.
To investigate whether localization outside of the visible
confines of chromosome territories can also occur for regions
that are not coordinately regulated, we have examined the
G
spatial organization of human 11p15.5 and the syntenic
region on mouse chromosome 7. This region is gene rich
but its genes are not coordinately expressed, rather overall
high levels of transcription occur in several cell types. We
found that chromatin from 11p15.5 frequently extends
away from the chromosome 11 territory. Localization outside
of territories was also detected for other regions of high
gene density and high levels of transcription. This is shown
to be partly dependent on ongoing transcription. We suggest
that local gene density and transcription, rather than the
activity of individual genes, influences the organization of
chromosomes in the nucleus.
Introduction
Human nuclei have a radial organization. Chromosomes
with the highest gene density are preferentially disposed toward
the nuclear interior, and gene-poor chromosomes locate
towards the nuclear periphery (Croft et al., 1999; Boyle et
al., 2001; Cremer et al., 2001). This organization is conserved
in other vertebrates (Habermann et al., 2001; Tanabe et al.,
2002), suggesting that the nuclear interior may facilitate, or
create a permissive environment for, transcription. However,
many human chromosomes are a patchwork of domains
with varying gene density and so some very gene-rich regions of
the human genome are contained on chromosomes located
close to the nuclear periphery.
We have previously shown that individual human genes
can be transcribed from within the interior of chromosome
territories that are not located in the nuclear center (Mahy et
al., 2002). This showed that genes do not need to be either
at the visible surface of interphase chromosome territories,
or at the centre of the nucleus, in order to be transcribed.
These genes were located in regions of moderate gene-density
(the R-band 11p13). In contrast, the gene-dense major histo-
compatibilty complex (MHC)* locus is frequently observed
on loops of chromatin that extend away from the human
chromosome 6 territory that is detected by FISH with a
chromosome paint, particularly when transcription of genes
from this region is induced (Volpi et al., 2000). Similarly,
the epidermal differentiation complex (EDC) at 1q21 is
frequently located outside of the chromosome 1 territory in
keratinocytes, cells in which the genes of the EDC are highly
expressed (Williams et al., 2002). It was not clear whether
localization outside of chromosome territories was a particular
feature of regions of the genome that contain genes with
related functions, and that are coordinately expressed, or
whether it might represent a more general facet of genome
organization wherever genes are particularly clustered together,
or where the overall levels of transcription from a large
number of genes across a region is high.
To address this, we have used FISH to examine territory
organization of regions of the human genome with high
Address correspondence to W.A. Bickmore, MRC Human Genetics
Unit, Crewe Road, Edinburgh, EH4 2XU, UK. Tel.: 44-131-332-2471.
Fax: 44-131-343-2620. E-mail: W.Bickmore@hgu.mrc.ac.uk
*Abbreviations used in this paper: ActD, Actinomycin D; DRB, 5,6-
dichloro-
?
-ribofuranosylbenzimidazole; EDC, epidermal differentiation
complex; HSA11p, Homo sapiens chromosome 11 p arm; MAA, methanol:
acetic acid; Mb, megabase; MHC, major histocompatibilty complex;
MMU, Mus musculus chromosome; pFa, para-Formaldehyde.
Key words: gene density; chromatin; chromosome territories; nuclear
organization; transcription

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Volume 159, Number 5, 2002
gene densities and generally high levels of transcription. The
T-band 11p15.5 contains at least 47 known genes within
the most distal 4.5 megabase (Mb) of DNA. We found that
many megabases of this chromatin is frequently found out-
side of the visible confines of the 11p territory. By extending
this observation to other gene-dense parts of the human ge-
nome including; 11q13 and 16p13.3 and gene-dense re-
gions of chromosomes 21 and 22, we suggest that there is a
correlation between domains of high gene density and local-
ization outside of chromosome territories. We show that the
frequency of extraterritory localization decreases, but is not
eliminated, when transcription is inhibited. This level of
higher-order genome organization is conserved in the
mouse, indicating that it likely has functional significance.
We suggest that the propagation of a decondensed chroma-
tin fibre outside of the confines of chromosome territories
creates an environment that is permissive to transcription in-
creasing the overall transcriptional potential of the domain
Figure 1.
region of conserved synteny from
MMU7. (Left) Map of the distal 6 Mb of
human HSA11p15 from the 11p telomere
(tel) encompassing 11p15.5 and
11p15.4 and extending down to
11p15.3, showing the relative position
of genes and loci used in this study.
Genes are indicated in italics. Asterisks
indicate the positions of loci used in this
study. (Right) Map of the region of
MMU7 in conserved synteny to the
BWS-associated region of human
11p15. Gene locations were taken
from NCBI online sequencing data
(http://www.ncbi.nlm.nih.gov/cgi-bin/
Entrez/maps.cgi?org?hum&chr?11),
and from published maps (Alders et al.,
1997; Hu et al., 1997; Reid et al., 1997;
Buettner et al., 1998; Bepler et al., 1999;
Kato et al., 1999; Lee et al., 1999;
Engemann et al., 2000; Onyango et al.,
2000; Paulsen et al., 2000).
Map of HSA11p15 and the

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The organization of chromosome territories |
Mahy et al. 755
(Tumbar et al., 1999), and that the structure of chromo-
some territories is, in part, driven by transcription.
Results
Distal 11p15.5 can locate outside of
the chromosome 11 territory
It has been suggested that genes are positioned at the surface
of chromosome territories (Kurz et al., 1996; for review
see Cremer and Cremer, 2001). However, we recently re-
ported that actively transcribing genes from the moderately
gene-rich band
Homo sapiens
(HSA11p)13, or the region of conserved synteny on mouse
chromosome (MMU)2 are located within the interior of the
chromosome territory (Mahy et al., 2002). Conversely, the
very gene-dense MHC locus is frequently observed on chro-
matin loops that extend away from the HSA6 chromosome
territory (Volpi et al., 2000). These data suggest that the hu-
chromosome 11 p arm
Figure 2.
11p15.4, or 11p15.5 (red) together with an HSA11p paint (green) to MAA-fixed lymphoblast (a) or primary fibroblast (b) nuclei. Known
genes are italicized. (c) Maximal intensity projection of image stacks after three-dimensional FISH with probes from 11p15.5 (red) and a
paint for 11p (green) on pFa-fixed fibroblast nuclei. Cells were counterstained with DAPI (blue). (d and e) Views of MAPaint reconstructions
of pFa fixed fibroblast nuclei after three-dimensional FISH with probes (red) for IGF2 (d) and 80N22 (e) together with paints (green) for
11p (d) and 11q (e). Bars, 5 ?m.
Visualizing the spatial organization of 11p15 relative to the 11p chromosome territory. FISH of selected probes from 11p15.3,

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Volume 159, Number 5, 2002
Table I.
The proportion of signals external to chromosome territories
Gene/locus (cytogenetic location)Percentage of signals outside of chromosome territory
Two-dimensional (MAA-fixed)Three-dimensional (pFa-fixed)
WAGR (11p13)
Wt1 (MMU2)
D11S12 (11p15.4)
D11S679 (11p15.5)
IGF2 (11p15.5)
D11S483 (11p15.5)
11p tel (11pter)
245N5 (MMU7)
80N2 (11q13)
MHC Class II (6p21.3) fibroblasts
MHC Class II (6p21.3) fibroblasts
MHC Class II (6p21.3) keratinocytes
MHC Class II (6p21.3) lymphoblasts
EDC (1q21) keratinocytes
EDC (1q21) lymphoblasts
11
3
43
68
72
88
87
40
69
13
26
19
34
25
8
1
ND
30
31
28
43
58
ND
57
12
19
12
36
22
6
?
IFN-
?

The proportion (%) of signals scored as outside of chromosome territories in two-dimensional FISH (MAA-fixed) and three-dimensional FISH (fixed in pFa).
All human chromosome 11 loci were analysed in fibroblasts. Mouse loci (MMU) were examined in embryonic stem cells. The analysis of the human MHC
Class II (6p21.3) and EDC (1q21) of Volpi et al. (2000) and Williams et al. (2002) in fibroblasts, keratinocytes, and lymphoblasts is shown for comparison.
For two-dimensional analysis,
n = 100; for three-dimensional analysis, n = 35.
man genome is subject to different constraints of spatial or-
ganization, and that gene density or density of transcribed
genes, rather than the activity of individual genes may influ-
ence chromosome organization.
To address this, we have analyzed the organization of
distal 11p15, including the very gene-rich, subtelomeric
T-band 11p15.5. The most distal 4.5 Mb of HSA11p is well
characterized due to its association with Beckwith-Wiede-
mann Syndrome, and because of the cluster of imprinted
genes located there. The region contains at least 47 known
genes (Redeker et al., 1994; Alders et al., 1997; Hu et al.,
1997; Reid et al., 1997; Lee et al., 1999; Engemann et al.,
2000; Onyango et al., 2000; Paulsen et al., 2000; Fig. 1). In
addition, it has a density of CpG islands that is much higher
than that of 11p13 (Craig and Bickmore, 1994).
Cosmids encompassing the most distal part of 11p
(11p15.3–p15.5), and a PAC to the 11p telomere itself,
were first cohybridized with an 11p paint to methanol:acetic
acid (MAA; 3:1 vol/vol)-fixed nuclei from lymphoblasts
(Fig. 2 a) and primary fibroblasts (Fig. 2 b). For each probe,
the mean distance (
?
m) between the probe signal and the
nearest chromosome territory edge was calculated as de-
scribed previously (Mahy et al., 2002). In contrast to the ex-
pressed RCN gene in 11p13, for which 76% of signals were
well within (
?
0.2
?
m from the territory edge) the 11p terri-
tory (Mahy et al., 2002), all 11p15 loci were closer to the
territory surface (Fig. 3 a). Only 48% of signals from probe
cq26 in 11p15.3 were well within the 11p territory, decreas-
ing to 32% of D11S12 (11p15.4) signals, 25% of signals
from the
IGF2 gene in 11p15.5, and only 11% of signals
from the 11p telomere. Moreover, for the more distal loci,
an increasing proportion of signals from these probes was
found outside of the limits of the territory detectable by
FISH with a chromosome paint. 53% of
located
?
0.2
?
m beyond the 11p territory in this experi-
IGF2 signals were
ment rising to 80% of signals from 11pter (Fig. 3 a). The
null hypothesis that the location of a distal 11p15.5 probe
(e.g., cI-11p15-25) was the same as cq26 in 11p15.3, was re-
jected using a 2 sample
t-test, P
has the same location as an adjacent probe cI-11p15-46,
P
?
0.43. Volpi et al. (2000) similarly observed a high pro-
portion of signals (up to 36%) from the MHC locus located
outside of the painted human chromosome 6 territory in
MAA-fixed lymphoblastoid nuclei, though the distance
from the territory edge was not established in that case. The
mean distance of 11p15 probes from the territory edge con-
firmed this trend (Fig. 3 b). All 11p15.4 probes analyzed lie
within the territory but close to the edge, whereas the mean
positions of 11p15.5 probes are outside of the chromosome
territory. The mean location of 11p15.5 distal markers is
?
1
?
m beyond the chromosome territory (Fig. 3 b).
MHC class II genes are more frequently observed outside
of chromosome 6 territories in expressing cells (lymphoblas-
toid) than in cells that do not express class II genes (e.g., ke-
ratinocytes). Conversely, the localization of the EDC com-
plex outside of chromosome 1 territories is more frequent in
keratinocytes than lymphoblasts (Table I) (Williams et al.,
2002). This suggested that localization outside of chromo-
some territories may relate to the levels of transcription ema-
nating from these regions. Unlike the MHC or EDC, the
genes in distal 11p15.5 are not functionally related nor is
there any evidence that they are coordinately regulated.
Analysis of the organization of this region in MAA-fixed pri-
mary fibroblasts gave a similar result to that seen in lympho-
blast nuclei (Fig. 3 b). Hence, localization of distal 11p15.5
outside of the chromosome territory is not specific to any
one cell type with a specific pattern of gene expression.
Our previous analyses of intraterritory organization of
11p13 gave similar results whether the analysis was carried
out on flattened specimens fixed in MAA or on three-

?
0.000, but cI-11p15-25

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The organization of chromosome territories |
Mahy et al. 757
Figure 3.
the 11p territory edge (?m) after FISH to MAA fixed lymphoblast nuclei. An 11p13 probe is shown for comparison. Negative distances
indicate probe signals located beyond the visible limits of the detectable chromosome territory. The localization of cI-11p15–25 is
significantly different from that of cq26 (P ? 0.000). (b) Mean distance (?95% confidence interval/CI) of 11p15 probes from the edge of
the 11p territory of MAA-fixed lymphoblast (?) and fibroblast (?) nuclei. The number of territories analyzed (n) ?100. The mean position
of a probe for the PAX6 gene in 11p13 (Mahy et al., 2002) and the chromosome 11 centromere (11pcen) are shown for comparison.
cI-11p15.25 and cI-11p15–46 have the same location outside of the 11p territory (P ? 0.43); however, cI-11p15–25 is significantly more
distant from the territory than cq26 (P ? 0.000). (c) Mean distance (?95% CI) in ?m of probes from the edge of the 11p territory of
pFa-fixed fibroblast nuclei (n ? 35).
Quantifying the spatial organization of 11p15. (a) Histogram showing the distribution of signals from 11p15 probes relative to