An epichromatin epitope: persistence in the cell cycle and conservation in evolution.
ABSTRACT Interphase nuclear architecture is disrupted and rapidly reformed with each cell division cycle. Successive cell generations exhibit a "memory" of this nuclear architecture, as well as for gene expression. Furthermore, many features of nuclear and mitotic chromosome structure are recognizably species and tissue specific. We wish to know what properties of the underlying chromatin structure may determine these conserved features of nuclear architecture. Employing a particular mouse autoimmune anti-nucleosome monoclonal antibody (PL2-6), combined with deconvolution immunofluorescence microscopy, we present evidence for a unique epitope (involving a ternary complex of histones H2A and H2B and DNA) which is localized only at the exterior chromatin surface of interphase nuclei and mitotic chromosomes in mammalian, invertebrate and plant systems. As only the surface chromatin region is identified with antibody PL2-6, we have assigned it the name "epichromatin". We describe an "epichromatin hypothesis", suggesting that epichromatin may have a unique evolutionary conserved conformation which facilitates interaction with the reforming post-mitotic nuclear envelope and a rapid return of interphase nuclear architecture.
[show abstract] [hide abstract]
ABSTRACT: Genomes are more than linear sequences. In vivo they exist as elaborate physical structures, and their functional properties are strongly determined by their cellular organization. I discuss here the functional relevance of spatial and temporal genome organization at three hierarchical levels: the organization of nuclear processes, the higher-order organization of the chromatin fiber, and the spatial arrangement of genomes within the cell nucleus. Recent insights into the cell biology of genomes have overturned long-held dogmas and have led to new models for many essential cellular processes, including gene expression and genome stability.Cell 03/2007; 128(4):787-800. · 32.40 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: In the limited space of the nucleus, chromatin is organized in a dynamic and non-random manner. Three ways of chromatin organization are compaction, formation of loops and localization within the nucleus. To study chromatin localization it is most convenient to use the nuclear envelope as a fixed viewpoint. Peripheral chromatin has both been described as silent chromatin, interacting with the nuclear lamina, and active chromatin, interacting with nuclear pore proteins. Current data indicate that the nuclear envelope is a reader as well as a writer of chromatin state, and that its influence is not limited to the nuclear periphery.FEBS Letters 07/2008; 582(14):2017-22. · 3.54 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: An increasing number of studies indicate that chromosomes are spatially organized in the interphase nucleus and that some genes tend to occupy characteristic zones of the nuclear volume. FISH studies in mammalian cells suggest a differential localization of active and inactive loci, with inactive heterochromatin being largely perinuclear. Recent genome-wide mapping techniques confirm that the nuclear lamina, which lies beneath the nuclear envelope, interacts preferentially with silent genes. To address the functional significance of spatial compartmentation, gain-of-function assays in which chromatin is targeted to the nuclear periphery have now been carried out. Such experiments yielded coherent models in yeast; however, conflicting results in mammalian cells leave it unclear whether these concepts apply to higher organisms. Nevertheless, the recent discovery that evolutionarily conserved inner nuclear membrane proteins support the peripheral anchoring of yeast heterochromatin suggests that certain principles of nuclear organization may hold true from yeast to man.Current opinion in genetics & development 04/2009; 19(2):180-6. · 8.99 Impact Factor
©2011 Landes Bioscience.
Do not distribute.
www.landesbioscience.com Nucleus 47
Nucleus 2:1, 47-60; January/February 2011; © 2011 Landes Bioscience
*Correspondence to: Donald E. Olins; Email: firstname.lastname@example.org
Submitted: 07/06/10; Revised: 09/15/10; Accepted: 09/16/10
The appearance of histones and nucleosomes in the earliest
eukaryotes laid the groundwork for an entirely new set of archi-
tectural principles for packaging of the DNA, not previously seen
in the evolution of life. Describing these principles is a major goal
of cell biology and encompasses understanding the interphase
nucleus, the mitotic chromosome and other chromatin structural
states.1-5 A major advance in our understanding of the organi-
zation of chromatin came with the description of chromosome
territories (CTs),6,7 which demonstrated that the individuality
of mitotic chromosomes is preserved, albeit in a swollen form,
within the interphase nucleus. Evidence has also been presented
that within these territories chromatin exists in “knot-free” glob-
ules, analogous to the nucleosomal “beads-on-a-string”; but at
Interphase nuclear architecture is disrupted and rapidly reformed with each cell division cycle. Successive cell generations
exhibit a “memory” of this nuclear architecture, as well as for gene expression. Furthermore, many features of nuclear
and mitotic chromosome structure are recognizably species and tissue specific. We wish to know what properties of the
underlying chromatin structure may determine these conserved features of nuclear architecture. Employing a particular
mouse autoimmune anti-nucleosome monoclonal antibody (PL2-6), combined with deconvolution immunofluorescence
microscopy, we present evidence for a unique epitope (involving a ternary complex of histones H2A and H2B and DNA)
which is localized only at the exterior chromatin surface of interphase nuclei and mitotic chromosomes in mammalian,
invertebrate and plant systems. As only the surface chromatin region is identified with antibody PL2-6, we have assigned
it the name “epichromatin”. We describe an “epichromatin hypothesis”, suggesting that epichromatin may have a unique
evolutionary conserved conformation which facilitates interaction with the reforming post-mitotic nuclear envelope and
a rapid return of interphase nuclear architecture.
An epichromatin epitope
Persistence in the cell cycle and conservation in evolution
Ada L. Olins,1 Markus Langhans,2 Marc Monestier,3 Andreas Schlotterer,4 David G. Robinson,2 Corrado Viotti,2
Hanswalter Zentgraf,5 Monika Zwerger6,† and Donald E. Olins1,*
1Department of Pharmaceutical Sciences; College of Pharmacy; University of New England; Portland, ME USA; 2Department of Cell Biology; Heidelberg Institute for Plant
Sciences; 4Department of Medicine I and Clinical Chemistry; University of Heidelberg; 5Applied Tumor Virology, Electronmicroscopy; 6“Functional Architecture of the Cell”;
Department of Molecular Genetics; German Cancer Research Center; Heidelberg, Germany; 3Temple Autoimmunity Center; Department of Microbiology and Immunology;
Temple University School of Medicine; Philadelphia, PA USA
†Current address: Brigham and Women’s Hospital/Harvard Medical School; Partners Research Facility; Cambridge, MA USA
Key words: interphase nuclear architecture, mitotic chromosomes, chromatin, epichromatin, evolution
Abbreviations: H2A, H2B, H3, H4, nucleosome inner histones; CREST, auto immune antibody against centromere proteins;
CENH3, centromere H3 variant; H3(S10)p, anti-phosphorylated serine 10 in H3; mAb, monoclonal antibody; SLE, systemic
lupus erythematosus; ELISA, enzyme-linked immunosorbent assay; CT, chromosome territory; LBR, lamin B receptor; ELCS,
nuclear envelope-limited chromatin sheets; DIC, differential interference contrast; NE, nuclear envelope; CB, Coomassie blue;
PAGE, polyacrylamide gel electrophoresis; ECL, enhanced chemiluminescence; LADs, lamina-associated domains;
TM, transmembrane domain; ER, endoplasmic reticulum; PFA, paraformaldehyde; DTT, dithiothreitol
a larger scale, encompassing megabase lengths of chromosomal
DNA.8 Compartmentalization of silenced heterochromatin to
the periphery of the interphase nucleus is a common architec-
tural theme. Current evidence supports that there are numerous
lamina-associated peripheral chromatin domains, characterized
by low gene-expression, which may facilitate global nuclear orga-
nization.9,10 To the extent that these architectural principles may
be universal among the eukaryotes, one must ask how they are
maintained through successive cell generations and during the
extensive evolution of new species.
Anti-nuclear antibodies are a frequent occurrence in the sera
of autoimmune humans and animals, being especially prevalent
in systemic lupus erythematosus (SLE).11,12 These autoantibodies
can be directed against a variety of nuclear antigens, including
DNA, histones, nucleosomes and subnucleosomal particles. As
©2011 Landes Bioscience.
Do not distribute.
48 Nucleus Volume 2 Issue 1
U2OS and HL-60/S4 cells by deconvolution immunofluores-
cence microscopy with the aim of searching for chromatin sub-
structures. One anti-nucleosome antibody (PL2-6) specifically
stained a chromatin compartment at the periphery of interphase
nuclei, as well as staining the surface of mitotic chromosomes.
We suggest that this compartment represents a unique surface
chromatin conformation, to which we assign the name “epichro-
matin”. Furthermore, we formulate an “epichromatin hypoth-
esis”, suggesting that this chromatin may play a crucial role in
organizing interphase nuclear architecture, justifying its conser-
vation in the evolution of cell structure.
Immunofluorescent analysis of a set of mouse anti-nucleosome
autoantibodies. A number of mAbs derived from autoimmune
mice were tested by immunostaining against rapidly growing
HL-60/S4 (suspension cells) and U2OS (attached cells). The
binding specificities of the mAbs (all IgGs) and verification that
they are all anti-nucleosome antibodies have previously been
determined by ELISA.11,13,14,17,18 Examples of the deconvolved
slices from selected antibodies are shown in Figure 1: HL-60/
S4 cells are displayed in A; U2OS cells in B. Monoclonal PL2-6
and PL2-7 were derived from the same mouse and are both anti-
H2A-H2B-DNA;14,17 LG10-1 is from a different animal and has
anti-H3-H4-DNA specificity.14,18 For the set of ten mAbs tested,
the most typical immunostained images resembled those pre-
sented for LG10-1 and PL2-7; i.e., staining throughout interphase
nuclei (except nucleoli) and throughout mitotic chromosomes,
paralleling the DAPI staining of DNA. With one mAb, PL2-6,
the images were quite different; i.e., staining was confined to
the periphery of interphase nuclei and metaphase chromosomes,
which we name “epichromatin”. By ELISA, PL2-6 resembled six
other anti-H2A-H2B-DNA autoantibodies (including PL2-7)
derived from the same animal.17 All displayed strong ELISA
reactivity with the adsorbed complex of H2A-H2B-DNA, less
reaction with H2A-H2B, and weak-to-negligible reactions with
adsorbed separate histones or with an adsorbed H3-H4-DNA
complex.17 One difference, observed by ELISA, is that the reac-
tivity of PL2-6 to adsorbed H2A-H2B-DNA could be inhibited
by preincubation with the H2A-H2B-DNA complex in buffer;
which was not the case with the other tested mAbs.14 The unique
staining pattern of PL2-6 (especially, the immunolocalization at
the periphery of mitotic chromosomes) provoked us to examine
accessibility of this epichromatin epitope throughout the cell
The epichromatin epitope is present throughout the cell
cycle. Immunostaining experiments clearly demonstrated that the
epichromatin epitope recognized by PL2-6 is present throughout
the cell cycle, even after nuclear envelope (NE) breakdown and
before post-mitotic NE reformation. Immunostaining images
throughout mitosis, plus interphase, are presented for U2OS
(Fig. 2A and Sup. Vid. 1–5) and for HL-60/S4 cells (Fig. 2B).
Identical results were obtained with formaldehyde (PFA) or with
methanol/acetone fixation; only the PFA results are presented in
Figure 2. During prophase the epichromatin epitope is accessible
part of a program to analyze the mechanisms underlying autoim-
mune disorders, several mouse strains have been identified that
spontaneously develop SLE and are the source of a number of
monoclonal anti-nucleosomal antibodies.13,14 We have employed
some of these mouse monoclonal antibodies (mAbs) to probe
nuclear structural domains. Historically, this approach was use-
ful in identifying the histone H3 variants of centromeric regions
employing CREST autoantibodies from scleroderma patients.15,16
We tested a panel of these mouse mAbs on rapidly growing
Figure 1. Immunostaining of mammalian tissue culture cells with se-
lected mouse monoclonal anti-nucleosome antibodies. Cell types:
(A) HL-60/S4; (B) U2OS. Mouse mAbs (PL2-6, PL2-7 and LG10-1) staining
are shown in red: DAPI staining in blue. The arrows denote mitotic cells.
The arrowheads point to prophase nuclei. Each image is a single decon-
volved optical slice. Bar equals 10 μm for both (A and B).
©2011 Landes Bioscience.
Do not distribute.
www.landesbioscience.com Nucleus 49
epichromatin epitope in mitotic chromosomes does not depend
upon close proximity of the chromatin to an intact NE or to
components of the NE. The epichromatin epitope appears to be
an inherent property of chromatin throughout the cell cycle.
The epichromatin epitope appears to be absent from sev-
eral nuclear and chromosomal sites during the cell cycle. As
described earlier, PL2-6 does not stain the internal chromatin
to antibody at peripheral regions of the condensing chro-
mosomes adjacent to the dissociating nuclear envelope.
During metaphase and early anaphase, the epitope out-
lines the exterior surface of the congressed chromosomes;
more internal chromosome arms and ends do not exhibit
the epitope. By late anaphase the epichromatin epitope
becomes visible on the internal trailing arms and ends of
some mitotic chromosomes. Finally, during telophase the
epichromatin epitope “spreads” around the nuclear periph-
ery, as the clustered mitotic chromosomes fuse and the NE
reforms. By contrast, co-immunostaining with the “mitotic
marker”, rabbit anti-H3(S10)p,19,20 clearly revealed the
presence of this antigenic determinant during mitosis and
its disappearance during interphase (Fig. 3B); whereas
the epichromatin epitope persisted at every phase of the
cell cycle (Fig. 3A). Examination of serial slices of mitotic
figures (data not shown) demonstrated that the H3(S10)
p antigenic determinant is present throughout the chromo-
some set, although staining is stronger near the periphery.
This observation (i.e., stronger peripheral staining) is in
agreement with previously published images (see figure 3
in ref. 19 and figure 1 in ref. 20). When the “merge” image
is examined closely (insert, Fig. 3D), the epichromatin epi-
tope appears to be slightly exterior to, and overlapping with
H3(S10)p. An additional co-immunostaining experiment
attempted to determine whether centromeric regions, as
detected with autoimmune CREST antisera, revealed any
special relationship to the epichromatin epitope. Figure
3E presents single optical slices of a metaphase plate and
an interphase nucleus. All of the deconvolved slices were
examined in this experiment (data not shown). Careful
examination revealed that a few centromeres were close
to the region of the epichromatin epitope, but the major-
ity revealed no obvious proximity. The absence of PL2-6
staining in telophase chromosome “cores” is discussed in
a later section.
The epichromatin epitope persists in metaphase chro-
mosomes even with nuclear envelope components dis-
persed into the cytoplasm. During the dissolution of the
NE at mitosis, all of its components move into the cyto-
plasm.21 Integral membrane proteins are retained in the
endoplasmic reticulum (ER) by virtue of their transmem-
brane domains (TM). Lamin B receptor (LBR) is an excel-
lent example of an inner membrane protein that persists in
the ER during mitosis, returning early to the post-mitotic
reforming NE.22 Figure 4A demonstrates in U2OS cells
that LBR is present in the interphase NE, but withdraws
into the ER during mitosis; whereas PL2-6 stains both the
interphase epichromatin and the exterior of the mitotic
chromosome cluster. Similar sets of images were obtained for
emerin (Fig. 4B) and SUN2 (Fig. 4C), both proteins possess-
ing TM domains. Lamin A, which lacks direct attachment to
the NE, is also dispersed into the mitotic cell cytoplasm (Fig.
4D). Similar image data for LBR and SUN2 have been obtained
comparing interphase HL-60/S4 with metaphase cells (data not
shown). It is clear from these experiments that “exposure” of the
Figure 2. Immunostaining of the epichromatin epitope through mitosis in
U2OS (A) and HL-60/S4 (B) cells. Mouse mAb PL2-6 staining is shown in red;
DAPI staining in blue. Each image is a single deconvolved optical slice. Bar
equals 10 μm for both (A and B). Projection videos for some of these images
of U2OS can be found in the Supplementary Video Files, specifically: late
prophase (Video 1); early anaphase (Video 2); late anaphase (Video 3); early
telophase (Video 4); late telophase (Video 5).
©2011 Landes Bioscience.
Do not distribute.
50 Nucleus Volume 2 Issue 1
in previous figures, mid-section optical slices of interphase nuclei
suggest that the epichromatin epitope is continuous at the NE.
However, Figure 6A presents evidence that is not an accurate
impression. This Figure presents a gallery of tangential slices (i.e.,
just under the NE) stained with PL2-6, within interphase nuclei
of U2OS and HL-60/S4 cells. It is evident that the epichroma-
tin epitope exhibits a punctate/granular pattern at the NE. This
granular substructure at the NE is preserved in detergent-buffer
isolated nuclei from HL-60/S4 (Fig. 6B), washed with buffers
containing divalent cations (1.5 mM MgCl2) or treated with
combined chelators (EDTA and EGTA), prior to fixation and
immunostaining. Because this granular substructure is seen at
the NE of intact cells, it is unlikely to be an artifact of nuclear iso-
lation. As with the regions of interphase chromatin and mitotic
chromosomes that do not exhibit the epichromatin epitope,
described earlier, the staining substructure at the NE might arise
from a variety of possible causes which remain to be completely
elucidated. But it is clear that since central optical sections in iso-
lated and permeabilized nuclei show epichromatin staining only
at the nuclear periphery, this characteristic staining pattern is not
a consequence of inadequate antibody penetration.
Immunoelectron microscopy documents epichromatin epi-
tope localization at the nuclear envelope and in envelope-lim-
ited chromatin sheets (ELCS) of granulocytic HL-60/S4 cells.
When human leukemic HL-60 cells are treated with all-trans
retinoic acid (RA), they cease dividing and terminally differen-
tiate into granulocytic forms. This differentiation process takes
3–4 days in the subline HL-60/S4, leading to nuclear lobula-
tion and formation of extensive sheets containing a single layer of
~30 nm chromatin fibers sandwiched between apposed nuclear
envelopes.5,30 These envelope-limited chromatin sheets (ELCS)
are an extension of the adjacent NE membranes, with the ~30
nm chromatin layer in ELCS continuous to the most periph-
eral chromatin layer in the adjacent nuclear lobule. Employing
pre-embedded thin section immunoelectron microscopy with a
gold-labeled secondary antibody, the presence of the epichroma-
tin epitope can be clearly visualized (Fig. 7). Two examples of
HL-60/S4 granulocytic cells exhibiting nuclear irregularity and
extensive ELCS formation are presented in Figure 7A and B. C
of interphase nuclei or the “internal” chromosome arms and
ends of prophase, metaphase and early anaphase mitotic chro-
mosomes. Another location that appears unreactive to the anti-
epichromatin antibody is the mitotic chromosome “core”, a
region facing the spindle pole, possessing centromeres, binding
to the spindle microtubules, exhibiting proteins such as LAP2α
and BAF and excluding LBR, LAP2β and lamin B.23,24 Examples
of mitotic chromosome cores are presented in Figure 5A and
Supplementary Figure 1. Note especially that CREST antibody
stains centromere regions of the cores, while anti-LBR and PL2-6
primarily react with the telophase chromosome “periphery”. In
contrast, SUN2 appears to stain the core and periphery of telo-
phase chromosomes (Sup. Fig. 1). Eventually the daughter inter-
phase nuclei do show LBR and epichromatin staining around the
entire nuclear surface.
Within interphase nuclei of various types of attached cells
(e.g., HeLa, NRK, 3T3 and CHO), intranuclear channels or
tubules can be readily observed.25,26 These intranuclear tubules
are generally oriented perpendicular to the cell attachment sur-
face. Figure 5B and Supplementary Figure 2 present several
examples of U2OS cells with intranuclear tubules. Since the
tubules are primarily vertical in orientation, they usually present
circular cross-sections. Figure 5B demonstrates that these tubules
can be visualized by immunostaining with anti-LBR, but not
with anti-epichromatin. Supplementary Figure 2 demonstrates
that lamin A, emerin and SUN2 yield staining of the tubules, but
PL2-6 does not. Current evidence supports that these tubules are
extensions of the nuclear envelope, frequently adjacent to nucleoli
and involved in Calcium release.27-29
There are numerous reasons why selected regions of inter-
phase nuclei and mitotic chromosomes may not exhibit reactiv-
ity with the anti-epichromatin antibody. It may be that the local
chromatin regions do not have the epitope or that it is blocked by
bound molecules. Given that these regions can all be visualized
by other IgG antibodies of the same molecular size, but different
specificity, it is considered unlikely that there is a general penetra-
The epichromatin epitope exhibits a granular substructure
at the nuclear envelope in cells and in isolated nuclei. As shown
Figure 3. Co-immunostaining of U2OS interphase and metaphase cells with the epichromatin, mitotic marker and centromere (CREST) antibodies. The
mitotic cell is on top of each part; the interphase cell is at the bottom. (A) mouse mAb PL2-6; (B) rabbit anti-H3 phosphorylated at serine 10, the mitotic
marker H3(S10)p; (C) DAPI; (D) merge with an insert showing a 3-fold enlargement of the region denoted by an asterisk; (E) merged image of PL2-6,
CREST and DAPI. Mouse mAb PL2-6 staining is shown in red, H3(S10)p and CREST in green, DAPI in blue. Each part is a single deconvolved optical slice
of the same field. Bar equals 10 μm.
©2011 Landes Bioscience.
Do not distribute.
www.landesbioscience.com Nucleus 51
shows an enlargement of region “c” in A, with two ELCS lying
side-by-side; each is studded with gold grains. D presents an
enlargement (region “d“ of A) at the interphase NE, again
revealing gold-labeled antibody at the periphery. E shows a
single chromatin layer sandwiched in between the nucleolus
and the NE (region “e” of B) with clear labeling of the epi-
chromatin epitope. These images demonstrate that the epi-
chromatin epitope is present at the most peripheral single layer
of ~30 nm chromatin fibers and not detected within more
The NE and ELCS membranes can not be visualized in
Figure 7 for several reasons: (1) the post-fixation extraction
with 0.1% Triton X-100 removes most of the lipids; (2) the
samples were not fixed with OsO4, (which enhances lipid
contrast in the electron microscope). Evidence is presented
in Supplementary Figure 3 to show that an integral mem-
brane protein component of the NE (LBR) does remain in
place, even though the membrane lipids can not be visual-
ized. Immunoelectron microscopic staining of granulocytic
HL-60/S4 cells for LBR clearly demonstrates the presence of
gold particles along the ELCS and at the NE.
The epichromatin epitope is conserved among animals
and plants. In order to explore the evolutionary conservation
of the epichromatin epitope, studies were performed on a vari-
ety of eukaryotic organisms with well studied genetic systems.
The invertebrates, Drosophila melanogaster and Caenorhabditis
elegans, both have highly diverged homologs of vertebrate lam-
ins in their NE.31 Figure 8 presents image data to demonstrate
that the epichromatin epitope can be found at the periphery
of interphase nuclei in both species, as well as at the surface
of mitotic chromosomes in Drosophila Kc cells. This conclu-
sion is clearly shown in Figure 8A, which presents a merged
image of staining by mAb PL2-6 (red), rabbit anti-H3(S10)
p (green) and DAPI (blue). The 3x enlarged cluster of mitotic
chromosomes closely resembles a similar mitotic cluster shown
previously (Fig. 3) for U2OS cells; the epichromatin epitope
appears to be more exterior than the H3(S10)p determinant.
Figure 8B shows a portion of a Drosophila larval ovary. The
characteristic nuclear peripheral staining by PL2-6 can be
readily observed in the surface cells of the ovary, demonstrat-
ing the expected location of this epitope. However, deeper cells
within the ovary did not stain. We suspect that this is an anti-
body “penetration” problem due to the thickness of the whole
ovary. Figure 8C presents a PL2-6 immunostained image and
a DIC image of a C. elegans worm. The epichromatin epitope
can be detected at the periphery of the interphase nuclei. As
with the Drosophila larval ovary, antibody “penetration” into
the whole organism may have prevented the staining of more
internal cells. None-the-less, it is clear that the epichromatin
epitope staining of peripheral chromatin within interphase
nuclei exists in multicellular invertebrates, despite their highly
divergent NE composition.31
Plant cell NEs are even more divergent from higher meta-
zoans than observed with the invertebrates cited above, exhib-
iting an absence of homologs to lamins, LBR and most other
NE-associated proteins.32-34 None-the-less, Figure 9A–D
Figure 4. Dispersion of LBR, emerin and SUN2 into the mitotic ER and of
lamin A (LMNA) into the cytoplasm of U2OS cells, concurrent with persis-
tence of the epichromatin epitope at the periphery of mitotic chromo-
somes. Mouse mAb PL2-6 staining is shown in red. Anti-LBR (A), anti-emer-
in (B), anti-SUN2 (C) and anti-LMNA (D) staining are indicated in green; DAPI
in blue. In each part, the upper row of images is from the same mitotic cell;
the bottom row is from the same interphase cell. Bar equals 10 μm.
©2011 Landes Bioscience.
Do not distribute.
52 Nucleus Volume 2 Issue 1
study, we attempted to see whether PL2-6, PL2-7
and LG10-1 were capable of providing information
by immunoblotting procedures. Figure 10A pres-
ents an immunoblot analysis of PL2-6 reacted with
a total cell extract of U2OS cells (a similar experi-
ment with PL2-7 and LG10-1 did not provide any
ECL signals using the same extract of U2OS cells).
Figure 10A reveals that most of the extracted pro-
teins, when stained with Coomassie Blue (lane 2),
migrated between ~36 to ~100 kDa. However, the
major anti-epichromatin reactive band migrated at
~18 kDa (lane 3), a region which includes the inner
histones. A few very faint higher molecular weight
bands were also detected with PL2-6. Figure 10B
presents immunoblots of PL2-6 against several types
of samples, including core mononucleosomes from
HeLa cells and purified Xenopus recombinant core
histones, individually or in various equimolar com-
binations. The image of the immunoblot shown
in Figure 10B presents alternating lanes of the
Coomassie Blue (CB) stained membrane (lanes 1,
2, 4 and 6) interspersed with carefully aligned ECL
images from the same membrane, revealing PL2-6
reactivity (lanes 3, 5 and 7). Figure 10B lanes are as
follows: lane 1, CB stained protein molecular weight
markers; the region between lanes 1 and 2, repre-
sentation of the positions of the core histones (from
top to bottom, H3, H2B, H2A, and H4); lanes 2
and 3, HeLa mononucleosomes; lanes 4 and 5, equi-
molar mixture of recombinant Xenopus histones
H4, H2A, H2B and H3; lanes 6 and 7, equimolar
mixture of recombinant Xenopus histones H2A
and H2B. Even with the limited resolution of this
17.5% polyacrylamide gel, the data clearly indicate
that H4 and H3 show little reactivity with PL2-
6; but H2A and H2B appear to exhibit significant
reactivity. These results provoked us to perform dot
blots with the purified individual recombinant Xenopus inner
histones (Fig. 10C). Equimolar aliquots, based upon measure-
ments of the histone concentrations using the molar extinction
coefficients at 276 nm, were spotted onto Immobilon-P mem-
branes, reacted with PL2-6, analyzed by ECL (Fig. 10C, strip 1)
and CB stained (strip 2). The ECL results clearly show that,
on a molar basis, PL2-6 reacts most strongly with recombinant
Xenopus H2B and H2A and much more weakly with H4 and
H3. Semi-quantitative estimates of reaction intensities were
obtained from replicate dot arrays and different ECL exposure
times, employing ImageJ for measurement of film densities (see
Materials and Methods). The averaged results indicated that (set-
ting the H2B reaction intensity at 100%) H2A exhibited ~85%
reaction intensity, H4 ~22% and H3 ~9%. In summary, these
immunoblot results indicate that PL2-6 can recognize a homol-
ogous epitope within both histones H2A and H2B. Thus, the
anti-epichromatin antibody (PL2-6), which has been extensively
characterized by ELISA analyses, is capable of reacting by immu-
noblotting with the separate histones H2A and H2B, despite the
convincingly demonstrates that the epichromatin epitope is
present at the periphery of interphase nuclei and mitotic chro-
mosomes in tobacco BY-2 tissue culture cells and in interphase
nuclei of Arabidopsis thaliana root tips (Fig. 9E). Figure 9F dis-
plays an immunoelectron micrograph with gold-labeled anti-
body specifically localizing PL2-6 proximal to the NE in high
pressure freezing/freeze substitution post-embedded samples of
Arabidopsis thaliana root tips. Collectively, the immunostaining
of invertebrate animal and plant cells strongly argues that the epi-
chromatin epitope is highly conserved among very diverse species
with vastly different NE composition and, likely, very different
DNA sequences proximal to the NE.
Immunoblotting with PL2-6. Most of our current knowl-
edge about the binding specificity of the epichromatin anti-
body (PL2-6) is derived from ELISA studies.11,13,14,17,18 We know,
based upon ELISA quantitation, that PL2-6 binds strongly to
mononucleosomes and to a ternary complex of histones H2A +
H2B + DNA, weakly to H2A + H2B and very weakly to H3 +
H4 + DNA, individual histones or DNA alone. In the present
Figure 5. Nuclear and chromosomal regions that demonstrate an absence of epichro-
matin staining. (A) presents telophase U2OS cells with discernable chromosome “core”
regions. The left column of images pairs anti-centrosome CREST (top row) or anti-LBR
(second row) with DAPI. The middle column of images pairs PL2-6 with DAPI. The right
column presents a differential interference contrast (DIC) image of the separating
daughter cells in the same field. Arrows point to mitotic chromosome “cores”.
(B) displays interphase U2OS nuclei with intranuclear tubules. The left image pairs
anti-LBR with DAPI. The right image pairs PL2-6 with DAPI. Intranuclear tubules are
clearly stained by anti-LBR, but not by PL2-6. Bar equals 10 μm.
©2011 Landes Bioscience.
Do not distribute.
www.landesbioscience.com Nucleus 53
immunoblotting results with both native Hela and recombi-
nant H2A and H2B. The fact that PL2-6 reacts with individual
recombinant H2A and H2B argues that histone post-transla-
tional modifications are not a part of the epichromatin epitope.39
Furthermore, the data imply either that there are homologous
sites in the histone globular domains for Xenopus H2A and
H2B, similar enough to react with PL2-6 and/or sequences
within H2A and H2B that are each part of the conformational
epitope. Furthermore, these sequences should be present within
the corresponding mouse histones (i.e., the presumed immuno-
gens which induced the autoimmune antibody). A CLUSTAL
fact that these separate histones give very weak
reactions with PL2-6 by ELISA.
A chromatin epitope involving histones H2A
and H2B and DNA, recognized by a mouse
antibody (PL2-6), specifically localizes at the
peripheral surface of interphase chromatin and
mitotic chromosomes and is conserved by evo-
lution in its location among various plant and
animal cells. We suggest the name “epichroma-
tin” to denote this unique chromatin surface
conformation. To the best of our knowledge, no
such epitope has been reported before in either
interphase or mitotic cells, nor has there been
a suggestion that surface chromatin confor-
mation throughout the cell cycle differs from
bulk chromatin conformation. The staining of
interphase nuclei is reminiscent of the classical
“rim” pattern, commonly seen with antibod-
ies against lamins or nuclear envelope integral
membrane proteins (see for example, ref. 35 and
36). During mitosis the lamina/nuclear enve-
lope “rim” staining pattern disappears, being
last observed in early prophase and again vis-
ible at telophase, the periods when the nuclear
envelope is breaking down or reforming.21,37
By contrast, the epichromatin epitope persists
throughout mitosis, including metaphase, being
independent of the presence of a NE. In addi-
tion, we have shown that two closely related
mouse monoclonal autoimmune anti-nucleo-
some antibodies, PL2-7 and LG10-1 stain
throughout the entire interphase nuclei and
the entire set of mitotic chromosomes (gener-
ally resembling DAPI staining), minimizing the
possibility that the surface staining of PL2-6 is
due to a penetration problem.
Published and present data imply some
molecular characteristics about PL2-6 . ELISA
data11,13,14,17,18 demonstrate that the chromatin
epitope recognized by PL2-6 is a conforma-
tional one, involving a ternary complex of H2A,
H2B and DNA. Even though the epitope is present in the binary
complex of H2A and H2B, reactivity is augmented by complex-
ing with (mixed sequence) DNA on a surface, as in ELISA plates,
or in solution. Another observation (MM unpublished), using
a protocol employed with a different antibody,38 is that trypsin
removal of the histone basic tails in nucleosomes does not elimi-
nate the ELISA reactivity of the epitope. This implies that the
epichromatin epitope is present in the conserved globular regions
of histones H2A and H2B, which primarily reside within the
nucleosome cores. Even though the ELISA reactivity of PL2-6
with uncomplexed H2A or H2B is very weak,17 it is able to yield
Figure 6. Immunostaining of the epichromatin epitope in tangential optical sections of nu-
clei from U2OS and HL-60/S4 cells. Mouse mAb PL2-6 staining is shown in red; DAPI in blue.
(A) presents tangential sections of nuclei within intact cells. The top row of three images
displays sections of U2OS cells; the second row is from HL-60/S4 cells. (B) shows central and
tangential sections of isolated HL-60/S4 cell nuclei, washed in different buffers prior to fixa-
tion and immunostaining. In the top row, the isolated nucleus was washed in 1.5 mM MgCl2,
0.2 mM EGTA, 50 mM HEPES (pH 7.0); bottom row, washed in 0.2 mM EDTA, 0.2 mM EGTA,
50 mM HEPES (pH 7.0). Each image is a single deconvolved optical slice. In order to visualize
the low amount of epichromatin immunofluorescence at the tangent of the NE, the bright-
ness of the PL2-6 red signal was greatly increased. Bar equals 10 μm.
©2011 Landes Bioscience.
Do not distribute.
54 Nucleus Volume 2 Issue 1
(Fig. 5 and Sup. Fig. 1) that during late anaphase-telophase, the
mitotic chromosome “core” (centromere) region is unreactive
with PL2-6. There is increasing evidence that centromere regions
in a variety of species differ considerably in histone composition
and nucleosome conformation.41 Defining the molecular struc-
ture of the epichromatin epitope and possible binding interac-
tions remains a worthy endeavor.
It is tempting to speculate whether epichromatin may have a
functional significance, and if so, what it might be. At the onset
of such speculative thinking, it should be explicitly stated that
epichromatin may have no functional significance. It may simply
be that the epichromatin nucleosomal conformation results from
the lack of a complete surrounding by adjacent chromatin with
multiple alignment of the major mouse and Xenopus histones
(data not shown) does indicate scattered residue identities, con-
servative and neutral replacements within the histone globular
regions which are candidates for homologous epitope regions.
Although not yet established, it appears likely that most of the
nucleosomes within interphase and metaphase chromatin pos-
sess the histone amino acid residues involved in the epichromatin
epitope, but that these residues are not exposed (possibly due to
binding interactions or chromatin higher order structures). In
addition, there may be a subset of nucleosomes which have a dif-
ferent histone composition and lack the epichromatin epitope.
For example, mammalian centromeric regions contain H2A.Z in
many of the nucleosomes.40 Indeed, the present study documents
Figure 7. Immunoelectron microscopic labeling of the epichromatin epitope at the NE periphery and within ELCS of RA treated HL-60/S4 cells. (A and
B) display two different cells which exhibit nuclear lobulation and extensive formation of ELCS. Enlarged regions taken from (A and B) are as follows:
(C) taken from (A) (region “c“), presents two parallel ELCS; (D) taken from (A) (region “d“), displays a segment of the nuclear surface; (E) taken from
(B) (region “e“), shows a single peripheral heterochromatin layer adjacent to a nucleolus. The NE and ELCS membranes can not be visualized because
of the post-fixation detergent extraction and because the samples were not fixed with OsO4. Magnification bar values: (A and B) 1 μm; (C–E) 100 nm.
©2011 Landes Bioscience.
Do not distribute.
www.landesbioscience.com Nucleus 55
include specific variants that might preserve a unique chromatin
conformation by epigenetic mechanisms.
In many respects, our view of interphase epichromatin resembles
that of van Steensel and co-workers.2,9,10,48 This group has examined
the interphase nuclear chromatin regions that are proximal to the
nuclear envelope in Drosophila Kc cells and in human fibroblasts,
employing transfection with a chimeric gene containing lamin B
fused to DNA methylase, followed by subsequent analysis of the
methylated DNA fragments. These lamina-associated domains
(LADs) of chromatin do not display specific DNA sequences.
consequent exposure to cytoplasm (mitosis) or the nuclear
envelope (interphase). A physical analogy might be the sur-
face tension of liquid water. From this point-of-view, the
epichromatin nucleosomal conformation is the consequence
of a different macromolecular environment. Extending this
analogy, it is conceivable that epichromatin has a general
function; i.e., separating or protecting chromatin, creating a
barrier without membranes.
An alternative view is that epichromatin has a more spe-
cific functional significance, of sufficient importance to
be highly conserved in evolution. We describe this view as
the “epichromatin hypothesis”. This hypothesis postulates
that a unique peripheral chromatin conformation plays
an active role in ensuring the continuity of nuclear archi-
tecture throughout the cell cycle, especially during post-
mitotic nuclear envelope reformation. Furthermore, it is
suggested that this function (maintaining nuclear architec-
ture throughout the cell cycle) has been highly conserved by
evolution. Epichromatin could affect post-mitotic nuclear
reformation by presenting preferred interaction sites for
early-binding nuclear envelope integral membrane proteins
(e.g., LBR and LAP2β21,22,37). In a “deterministic” version of
the epichromatin hypothesis, this attractive chromatin con-
formation is always located at the same regions of specific
chromosomes, ensuring that these chromosome regions have
preferred positions adjacent to the nuclear envelope during
post-mitotic nuclear reformation. In a “stochastic” version
of the hypothesis, the epichromatin conformation does not
have precise chromosomal locations, but dynamically fluctu-
ates around chromosome surfaces. It is also conceivable that
the epichromatin regions exhibit tissue specificity, possibly
reflecting which regions of the genome are heterochromatic
or modified. We do not know whether the epichromatin epi-
tope is associated with specific DNA sequences; however, its
similar localization in divergent plant and animal cell nuclei
strongly suggests that neither specific DNA sequences nor
CpG DNA methylation42 are required.
There is at least one other nuclear structure, where spe-
cific DNA sequences do not directly dictate the formation
and location of an essential chromosomal feature; i.e., cen-
tromeres, which may represent a provocative comparison
to epichromatin. Centromeres are ubiquitous structures,
observed in plant and animal cells, involved in holding sister
chromatids together, being a platform for kinetochores and
ensuring proper segregation of homologous chromosomes
during mitosis.41,43-47 The underlying DNA sequence is vari-
able, comparing different species; although proximity to repetitive
DNA is a frequent motif. The current view is that centromeres
are epigenetic structures built around a centromere-specific highly
conserved histone H3 variant CENH3, which is critical for the
binding of other centromere-specific proteins and the establish-
ment of unique nucleosomal structures. CENH3 deposition on
chromatin does not occur during S phase; but, rather, at the end
of mitosis. Current studies are exploring the structural basis of the
epigenetic maintenance of centromere localization through cell
division. Clearly, we need to know whether epichromatin histones
Figure 8. Immunostaining of the epichromatin epitope in Drosophila
melanogaster and C. elegans cells. (A) Drosophila Kc cells immunostained
with mAb PL2-6 (red), rabbit anti-H3 phosphorylated at serine10, the mitotic
marker H3(S10)p (green) and DAPI (blue). (A) (right) is a 3-fold enlargement
of the mitotic chromosomes in (A) (left). (B) (left) displays PL2-6 staining (red)
of Drosophila ovary cells; DAPI (blue) of the same field is shown at (B) (right).
(C): C. elegans worm: left, immunostaining with PL2-6; right, DIC image.
Magnification bars equal 10 μm in (A and C); 5 μm, (B).
©2011 Landes Bioscience.
Do not distribute.
56 Nucleus Volume 2 Issue 1
It is possible that epichromatin includes (or is equivalent to) the
LADs within the interphase nucleus. Unfortunately, since the
nuclear envelope breaks down during mitosis, LADs can not be
analyzed from this stage of the cell cycle.
The observation that interphase CTs sometimes exhibit a
non-random radial distribution within interphase nuclei6,7,49 sug-
gests that for some CTs, epichromatin regions may participate in
directing chromosomes to proximity with the nuclear envelope.
However, our images from late anaphase and telophase cells (Fig.
2 and Suppl. Videos 3 and 4) suggest that late in mitosis most
chromosomes exhibit the surface epichromatin epitope. From the
They vary in size (0.1–10 Mb), are largely “gene-poor”, with those
genes present being transcriptionally repressed. Furthermore, there
is no simple correlation with epigenetic repressive histone markers
or with active histone markers; i.e., H3 and H4 acetylation and
H3K4 methylation are largely depleted. Perhaps the most revealing
characteristic of LADs occurs at their borders, which appear to be
relatively sharp, containing binding sites for an “insulator” protein
(CTCF), CpG islands and promoters of genes with transcription
directed away from the nuclear envelope. The authors suggest a
model with active gene loops interspersed between the LADs and
directed away from the repressive nuclear envelope environment.
Figure 9. Immunostaining of the epichromatin epitope in tobacco and Arabidopsis thaliana cells. (A–D), confocal sections of mitotic stages seen in to-
bacco BY-2 cells immunostained with mAb PL2-6 (red): (A) interphase; (B) metaphase plate; (C) anaphase; (D) telophase. (E) confocal section of a whole
mount of a Arabidopsis root tip stained with PL2-6 (red). (F) electron micrograph of a post-embedded immunogold stained thin section of a high
pressure freezing/freeze substituted Arabidopsis root tip. The arrows point to the 5 nm gold near the NE. The astericks indicate the position of nuclear
pores. CW, cell wall; C, cytoplasm; N, nucleus. Magnifications: (A–D), bar in (D) equals 10 μm; (E) bar equals 10 μm; (F) bar equals 200 nm.
©2011 Landes Bioscience.
Do not distribute.
www.landesbioscience.com Nucleus 57
Our observation of an epichromatin epitope in interphase and
metaphase cells provokes a new question: what is the relationship
between epichromatin and heterochromatin or euchromatin?
The immunogold electron microscopy of RA treated (granulo-
cytic) HL-60/S4 cells (Fig. 7) clearly demonstrates the presence
of the epichromatin epitope within the condensed heterochro-
matic region adjacent to the nuclear envelope, as well as within
the single heterochromatic layer of ELCS.5 These images prove
that the epichromatin epitope does include peripheral heterochro-
matin. However, it is becoming increasingly clear that some tran-
scriptionally-active euchromatin can be localized near the nuclear
envelope.2,56 Experiments should be devised to test whether such
euchromatin regions also exhibit the epichromatin epitope. Sites of
DNA synthesis occur in different regions of the interphase nucleus
during progression through S phase.57-59 The most peripheral chro-
matin of the interphase nucleus appears to replicate during late S,
yielding immunofluorescent images of incorporated nucleotides
very similar to those of epichromatin staining, and containing
the “G band” gene-poor heterochromatin. However, in our stud-
ies epichromatin staining of metaphase chromosomes does not
resemble the alternating patterns of R (gene-rich) and G bands,
traditionally employed for chromosome karyotyping. The basis
for our more uniform staining of the exposed surfaces of mitotic
chromosomes remains to be explored. It is important to mention
that mitotic chromosomes are surrounded by a complex mixture of
proteins and RNPs, called the “perichromosomal layer”,60,61 which
point-of-view of our epichromatin hypothesis, this implies that
other factors might restrict the chromatin surfaces involved in
post-mitotic nuclear envelope reformation. The complexities of
measurements and interpretations of CT arrangements within
interphase nuclei have been discussed.7 Radial and neighboring
arrangements among CT are not generally fixed or simple, which
may signify that epichromatin can not have a strictly deter-
ministic influence upon interphase nuclear architecture. Many
factors, including nuclear envelope shape, flexibility and com-
position and interactions with cytoskeletal elements must play
a role in defining nuclear architecture.50-52 To cite one example,
pericentromeric heterochromatin regions of mouse granulocytes
frequently cluster adjacent to the nuclear envelope making nod-
ules into the cytoplasm.53 In this situation, it is evident that the
nuclear envelope integral membrane protein LBR is necessary
for the heterochromatin to exhibit a peripheral localization. In
another remarkable variation of interphase nuclear architecture,
heterochromatin in the retinal rod cells of nocturnal (but not
diurnal) mammals is “inverted” (clustered) into the middle of
nuclei, apparently to function in channeling the light.54 A some-
what similar rearranged heterochromatin nuclear structure phe-
notype is observed in the mutant blood granulocytes of humans
(Pelger-Huet anomaly) and mice (Ichthyosis) due to a deficiency
of LBR.55 All of these nuclear variations suggest that the epichro-
matin conformation may be only one of many factors involved in
specifying post-mitotic nuclear architecture.
Figure 10. Immunoblot and immunodot analysis of the reactivity of PL2-6. (A) 4–20% gradient SDS-PAGE immunoblot analysis of U2OS total cell ex-
tract. Lanes: 1, BioLab protein molecular weight (mol wt) standards, stained with Coomassie Blue (CB), indicating the mol wt (kDa) of several proteins;
2, total cell extract stained with CB; 3, ECL reaction with PL2-6. (B) 17.5% SDS-PAGE with the following lanes: 1, protein mol wt markers stained with CB;
2 and 3, HeLa core mononucleosomes; 4 and 5, equimolar mixture of recombinant Xenopus inner histones H4, H2A, H2B and H3; 6 and 7, equimolar
mixture of recombinant Xenopus inner histones H2A and H2B. All lanes are from the same gel. Lanes 1, 2, 4 and 6, CB stained. Lanes 3, 5 and 7, ECL ex-
posures carefully aligned to lanes 2, 4 and 6, respectively. Mol wt values (kDa) of the markers are indicated to the left of lane 1. The four thin horizontal
lines between lanes 1 and 2 denote the positions of the four inner histones, starting with the lowest band (H4) and progressing upward, H4, H2A, H2B
and H3. (C) Immunodot blots of equimolar aliquots of purified individual recombinant Xenopus inner histones (H4, H2A, H2B and H3). Strip 1, ECL reac-
tion with PL2-6. Strip 2, identical membrane strip after CB staining.
©2011 Landes Bioscience.
Do not distribute.
58 Nucleus Volume 2 Issue 1
experiments, HL-60/S4 and U2OS cells were fixed with anhy-
drous methanol (-20°C, 10 min), followed by acetone (-20°C, 10
min) and subsequent washes with PBS, prior to blocking.
Primary antibody dilutions were as follows: PL2-6, PL2-7 and
most other mAbs, 1:200 dilution (~8 μg/ml); LG10-1, 1:400
(~3 μg/ml). For some experiments, the “mitotic marker” rabbit
anti-H3(S10)p (Millipore, Billerica MA) was employed at a 1:200
dilution. Co-immunostaining was also performed with human
auto-immune anti-centromere (CREST) antisera, as described
previously.35 Secondary antibody dilutions (Alexa 568 and 488)
were all at 1:100. DAPI was included during incubation with the
secondary antibodies. Incubations were in a moist chamber (1
hr, 37°C). Slides were mounted in SloFade Antifade Kit (Life
Technologies Co., Carlsbad CA) using a #1.5 thickness square
coverslip and sealed with clear nail polish. Optical sections on
all animal cells were collected on a DeltaVision Core microscope
(Applied Precision Inc., Issaquah WA) using either a 40x, 60x
or a 100x objective as “RGB” images. Images were deconvolved
using the built in SoftWoRx software. Adobe Photoshop was
used to assemble figures, adjust the size of individual images and
bring color levels of individual panels to comparable intensities,
no changes in gamma were made and then “RGB” was changed
to “CMYK”. Videos of selected mitotic U2OS cells stained with
PL2-6 were calculated from 12 projections between ±30o in 10o
intervals around the vertical axis, employing Imaris software
(Bitplane Inc., Saint Paul MN). The movies should be opened
with a video player in the “loop” mode.
Ovaries were dissected in PBS from 4-day-old Drosophila
melanogaster wild-type Oregon R females, grown at 25°C on
standard food. They were fixed in 4% PFA/PBS on ice for 30 min
and washed in PBS. After blocking with 2% BSA/0.1% Triton
X-100/PBS for 1 hour, the samples were incubated with PL2-6
(1:200) overnight at 4°C in blocking buffer. Subsequently, the
ovaries were washed for 1 hr in blocking buffer and incubated
1 hr with Alexa 568 anti-mouse in blocking buffer and washed
again for 1 hr. The nuclei were stained with DAPI in PBS for 10
min. After staining, the ovaries were spread out on a glass slide in
mounting medium (80% glycerol/0.4% N-propyl-gallate/PBS),
a coverslip was applied and sealed with nail polish.
The immunostaining method employed with C. elegans had
the following modifications. The worms were pelleted from M9
buffer and fixed overnight at 4°C in 4% PFA/PBS. Following
washes in 0.5% Triton X-100/PBS, the pellet was permeabilized
with 0.5% Triton X-100/1% DTT/PBS for 2 hrs at 37°C and
repeated washes as before. The worms were incubated in block-
ing buffer (1% BSA/0.5% Triton X-100/PBS) for 1 hr at RT,
followed by the primary antibody (PL2-6, 1:200) in blocking
buffer overnight at RT. Following additional washes, the col-
lected worms were incubated with secondary antibody (Alexa
568 anti-mouse, 1:100) in 0.1% BSA/0.5% Triton X-100/PBS for
4 hrs at 37°C. After final washes in 0.5% Triton X-100/PBS, the
stained worms were placed on microscope slides and embedded
in Vectashield Hard Set Mounting Medium for Fluorescence.
Arabidopsis thaliana plants were fixed and processed as pre-
viously described.66 Tobacco BY-2 suspension culture cells were
fixed and processed as described67 with minor changes. One batch
may influence the exposure of the epichromatin epitope and the
existence of an epichromatin conformation. It is of particular inter-
est that the cell proliferation associated nuclear antigen (Ki-67),62
which localizes within interphase nucleoli, appears to bind to the
surface of metaphase and anaphase chromosomes. It may be that
epichromatin can act as a “platform” for the binding of proteins or
particles which require distribution to both daughter nuclei.
Much remains to be determined about epichromatin, for
example: the molecular structure of the epitope; the nature of
the chromatin conformation; correlations with underlying or
surrounding DNA; correlations with epigenetic markers; char-
acterization of binding partners. Furthermore, it remains to be
established whether the concept of epichromatin and the sug-
gested “epichromatin hypothesis” will be useful in advancing our
understanding of the structural and functional “memory” of the
Materials and Methods
Reagents and antibodies. Paraformaldehyde (PFA) was prepared
as an 8% solution in distilled water (pH 7–8) and stored in ali-
quots at -20°C. Poly-L-lysine (MW 150–300 KD, Catalogue #
P-1399) and Nuclei EZ Prep (Catalogue # NUC-101) were pur-
chased from Sigma-Aldrich (St. Louis MO). Complete Mini pro-
tease inhibitor was obtained from Roche Diagnostics (Germany).
All buffers were made from reagent grade components, with stocks
sterilized by autoclaving or membrane filtration. Fluorescent sec-
ondary goat antibodies, Alexa 568 and Alexa 488 conjugates
were obtained from Invitrogen GmbH (Darmstadt, Germany).
Cells, tissues and organisms. Animal materials. Human
HL-60/S4 suspension cells were maintained in RMPI 1640
medium, plus 10% heated fetal calf serum, 1% Pen/Strep and
2 mM L-glutamine, as described earlier.30 Human U2OS cells
were cultivated in DMEM medium, plus 20% fetal calf serum
and 2 mM L-glutamine. Drosophila melanogaster Kc 167 cells
were maintained at 25°C, in Schneider’s Drosophila Medium
supplemented with 10% heated fetal calf serum, in the presence
of Pen/Strep. HL-60/S4 cells were harvested for microscopy
at a concentration of ~106/mL; coverslip attached U2OS and
Drosophila Kc cells were used prior to confluence. Caenorhabditis
elegans was cultivated on nematode growth medium (NGM) agar
at 20°C as described previously.63
Plant materials. A stably transformed Arabidopsis thaliana
line64 was grown on Murashige and Skoog medium contain-
ing 0.5% agar. Roots were harvested 5 days after germination
for whole mount immunofluorescence. Suspension cultures of
BY-2 tobacco (Nicotiana tabacum) were cultivated as previously
described65 and analysed 3 days after subculturing.
Immunostaining and fluorescence microscopy. HL-60/S4
cells were allowed to settle onto fresh polylysine-coated micro-
scope slides for 30–60 min at RT in a moist chamber. The
attached HL-60/S4, U2OS and Drosophila Kc cells were fixed
with fresh 4% PFA/PBS for 15 min at RT. The following steps
included: 50 mM NH 4Cl (1 min), PBS washes (2 x 1 min),
0.1% Triton X-100 in PBS (20 min); PBS (3 x 5 min); block-
ing with 10% normal goat serum/PBS (30 min, RT). For some
©2011 Landes Bioscience.
Do not distribute.
www.landesbioscience.com Nucleus 59
The cells were heated to 95oC for 5 min, allowed to cool and
sheared by repeated passage through a 26G x 1/2'' hypodermic
needle. 10 μL of the cell lysate was loaded per lane on a BioRad
4–20% precast gradient SDS-PAGE gel. Protein transfer, block-
ing, immunology and the ECL reaction were exactly as described
previously,68 employing PL2-6 at a 1:2,000 dilution.
For an immunoblotting experiment with histones, three sam-
ples were examined by electrophoresis in a 17.5% SDS-PAGE:
(1) native HeLa core mononucleosomes; (2) an equimolar mix-
ture of Xenopus recombinant core histones H4, H2A, H2B and
H3; (3) an equimolar mixture of Xenopus recombinant core his-
tones H2A and H2B. Different loads of each sample were run
on the gel, spanning 1–5 μg histones per lane. Protein transfer,
blocking, immunology and the ECL reaction were exactly as
described previously,68 employing PL2-6 at a 1:2,000 dilution.
The dot blotting procedure followed a protocol from Millipore
describing its use on Immobilon-P membranes. Concentrations
of the individual recombinant Xenopus inner histones H4, H2A,
H2B and H3 were measured by absorbance at 276 nm in gua-
nidine HCl buffer and calculated from known molar extinction
coefficients. These histone stock solutions were diluted to 100
μM with the immunoblotting transfer buffer (20 mM sodium
borate, 1 mM EDTA, pH 8.8). Several loads (3, 6 and 9 μl) of
each histone were applied to the Immobilon-P membrane in a
grid-like pattern on top of a filter paper stack. The spots were
allowed to dry, than wetted again with methanol prior to reac-
tion with PL2-6 (1:2,000) and ECL, as employed for immu-
noblotting. For a semi-quantitative analysis of the ECL reaction
intensities of the dots, three replicate dot arrays were established
onto the Immobilon-P membrane and several ECL exposure
times were obtained. Employing ImageJ software (rsb.info.nih.
gov/ij/), integrated spot densities were measured, corrected for
adjacent blank areas, normalized to the strongest reacting histone
(H2B, set to 1.0) and averaged to yield relative reaction strengths.
A.L.O. and D.E.O. wish to express their gratitude to Harald
Herrmann and Peter Lichter for generously hosting us in their
laboratories at the German Cancer Research Center (DKFZ,
Heidelberg), during which time this work was performed.
Gratitude is also expressed to the Department of Biology, Bowdoin
College for encouraging us to continue scientific research. Cristina
Pallares-Cartes (DKFZ) kindly provided the Drosophila sam-
ples. In addition, A.L.O. and D.E.O. thank Kati Toth and Jörg
Langowski (DKFZ) and Andreas Ladurner (EMBL, Heidelberg)
for gifts of HeLa mononucleosomal histones and recombinant
Xenopus inner histones, and to Thorsten Kolb (DKFZ) for the gift
of guinea pig anti-lamin A. Thanks are also extended to the ever-
helpful team in the Herrmann group “Functional Architecture
of the Cell”; Michaela Hergt, Helga Kleiner, Tanja Lichtenstern,
Monika Mauermann and Dorothee Möller.
Supplementary materials can be found at:
of cells was fixed with 4% PFA for 1 h; the other one with 1%
glutaraldehyde for 15 min. Samples were washed with Sörensen
buffer instead of PBS. Samples were incubated at 4°C with first
antibody (PL2-6, 1:200). ALEXA-FLUORR conjugate 546 (Life
Technologies Co.,) was used as secondary antibody. Imaging was
performed with a Zeiss Axiovert LSM510 Meta CLSM using a
C-Apochromat 63x/1.2 W corr water immersion objective. For
the Metadetector, the main beam splitter (HFT) 488/543 was
used. Pinholes were adjusted to 1 Airy Unit.
Immunoelectron microscopy. Two types of gold-labeled
immunoelectron microscopy were employed: (1) pre-embedded
immunostaining of fixed and detergent extracted HL-60/S4; (2)
post-embedded immunostaining on thin sections of high pressure
freezing/freeze-substituted preparations of Arabidopsis root tips.
For the pre-embedded staining reaction, the procedure largely
resembled that used for immunofluorescent staining, with the
following modifications: (1) permeabilization of the fixed cells
with 0.1% Triton X-100/PBS was extended to 30 min at RT;
(2) 6 nm gold-labeled secondary antibody was incubated for 4 hrs
in a moist chamber at 37°C, followed by three 5 min washes with
PBS; (3) after the antibody reactions, the coverslips were fixed with
2.5% glutaraldehyde in 50 mM cacodylate buffer for 5 min at RT
and 25 min at 4°C; (4) coverslips were given three washes in 50 mM
cacodylate prior to dehydration and embedding in epon, by standard
procedures; (5) coverslips were removed from the epon in liquid N2.
For the post-embedded immunostaining on high pressure
freezing/freeze substituted preparations of Arabidopsis thaliana
root tips, six-day-old root tips were cut from the seedling, sub-
merged in 140 mM sucrose, 7 mM trehalose and 7 mM Tris buffer
(pH 6.6). Four to five submerged root tips were collected, trans-
ferred to planchettes (Wohlwend GmbH, Sennwald, Switzerland;
type 241 and 242) and frozen in a high pressure freezer (Bal-Tec
HPM010, Lichtenstein). Freeze substitution was performed in
a Leica EM AFS2 freeze substitution unit (Leica, Germany) in
9.9 ml dry acetone supplemented with 100–200 μl 20% uranyl
acetate in methanol (0.2–0.4% final) at -85°C for 16 hrs before
gradually warming up to -50°C over a 5 hr period. After wash-
ing with 100% ethanol for 60 min, the roots were infiltrated and
embedded in Lowicryl HM20 at -50°C (intermediate steps of 30,
50, 75% HM20 in ethanol, 1 hr each), and polymerized for 3
days with ultraviolet (UV) light in the freeze substitution appara-
tus. To increase sectioning quality, the blocks were then hardened
with UV light for another 4 hr at RT. Ultrathin sections were cut
on a Leica Ultracut S and incubated with the primary antibody
(PL2-6, 1:3) followed by incubation with 5 nm gold-coupled sec-
ondary antibody (BioCell GAR5, 1:50) in 1% BSA/PBS. Sections
were examined in a JEM1400 transmission electron microscope
(JEOL, Japan) operating at 80 kV. Micrographs were recorded
with a FastScan F214 digital camera (TVIPS, Germany).
Immunoblotting and dot blotting. In order to immunob-
lot a U2OS total extract, cells were washed in 0.1 mM EDTA/
PBS, trypsinized, counted with a hemocytometer and washed in
PBS containing both Complete (Roche Diagnostics) and PMSF
(Sigma Aldrich) protease inhibitors. Cells (3 x 106) were lysed in
300 μL of 1x Laemmli sample buffer (10% glycerol, 3% SDS,
62.5 mM Tris-HCl, 50 mM DTT and 0.05% bromphenol blue).
©2011 Landes Bioscience.
Do not distribute.
nuclear envelope. Curr Opin Plant Biol 2009; 12:752-9.
35. Olins AL, Olins DE. Cytoskeletal influences on nuclear
shape in granulocytic HL-60 cells. BMC Cell Biol
36. Olins AL, Hoang TV, Zwerger M, Herrmann H,
Zentgraf H, Noegel AA, et al. The LINC-less granulo-
cyte nucleus. Eur J Cell Biol 2009; 88:203-14.
37. Margalit A, Vlcek S, Gruenbaum Y, Foisner R.
Breaking and making of the nuclear envelope. J Cell
Biochem 2005; 95:454-65.
38. Losman JA, Fasy TM, Novick KE, Massa M, Monestier
M. Nucleosome-specific antibody from an autoim-
mune MRL/Mp-lpr/lpr mouse. Arthritis Rheum 1993;
39. Luger K, Rechsteiner TJ, Flaus AJ, Waye MM,
Richmond TJ. Characterization of nucleosome core
particles containing histone proteins made in bacteria.
J Mol Biol 1997; 272:301-11.
40. Greaves IK, Rangasamy D, Ridgway P, Tremethick DJ.
H2A.Z contributes to the unique 3D structure of the
centromere. Proc Natl Acad Sci USA 2007; 104:525-30.
41. Dalal Y, Bui M. Down the rabbit hole of centromere
assembly and dynamics. Curr Opin Cell Biol 2010;
42. Kunert N, Marhold J, Stanke J, Stach D, Lyko F. A
Dnmt2-like protein mediates DNA methylation in
Drosophila. Development 2003; 130:5083-90.
43. Mellone BG. Structural and temporal regulation of
centromeric chromatin. Biochem Cell Biol 2009;
44. Gieni RS, Chan GK, Hendzel MJ. Epigenetics regulate
centromere formation and kinetochore function. J Cell
Biochem 2008; 104:2027-39.
45. Silva MC, Jansen LE. At the right place at the right
time: novel CENP-A binding proteins shed light on
centromere assembly. Chromosoma 2009; 118:567-74.
46. Przewloka MR, Glover DM. The kinetochore and the
centromere: a working long distance relationship. Annu
Rev Genet 2009; 43:439-65.
60 Nucleus Volume 2 Issue 1
47. Dalal Y. Epigenetic specification of centromeres.
Biochem Cell Biol 2009; 87:273-82.
48. Pickersgill H, Kalverda B, de Wit E, Talhout W,
Fornerod M, van Steensel B. Characterization of the
Drosophila melanogaster genome at the nuclear lamina.
Nat Genet 2006; 38:1005-14.
49. Croft JA, Bridger JM, Boyle S, Perry P, Teague P,
Bickmore WA. Differences in the localization and mor-
phology of chromosomes in the human nucleus. J Cell
Biol 1999; 145:1119-31.
50. Webster M, Witkin KL, Cohen-Fix O. Sizing up
the nucleus: nuclear shape, size and nuclear-envelope
assembly. J Cell Sci 2009; 122:1477-86.
51. Burke B, Roux KJ. Nuclei take a position: managing
nuclear location. Dev Cell 2009; 17:587-97.
52. Mejat A, Misteli T. LINC complexes in health and
disease. Nucleus 2009; 1:13.
53. Zwerger M, Herrmann H, Gaines P, Olins AL, Olins
DE. Granulocytic nuclear differentiation of lamin B
receptor-deficient mouse EPRO cells. Exp Hematol
54. Solovei I, Kreysing M, Lanctot C, Kosem S, Peichl L,
Cremer T, et al. Nuclear architecture of rod photore-
ceptor cells adapts to vision in mammalian evolution.
Cell 2009; 137:356-68.
55. Hoffmann K, Sperling K, Olins AL, Olins DE. The
granulocyte nucleus and lamin B receptor: avoiding the
ovoid. Chromosoma 2007; 116:227-35.
56. Akhtar A, Gasser SM. The nuclear envelope and tran-
scriptional control. Nat Rev Genet 2007; 8:507-17.
57. O’Keefe RT, Henderson SC, Spector DL. Dynamic
organization of DNA replication in mammalian cell
nuclei: spatially and temporally defined replication of
chromosome-specific alpha-satellite DNA sequences. J
Cell Biol 1992; 116:1095-110.
58. Ferreira J, Paolella G, Ramos C, Lamond AI. Spatial
organization of large-scale chromatin domains in the
nucleus: a magnified view of single chromosome ter-
ritories. J Cell Biol 1997; 139:1597-610.
59. Takebayashi S, Sugimura K, Saito T, Sato C, Fukushima
Y, Taguchi H, et al. Regulation of replication at the
R/G chromosomal band boundary and pericentromeric
heterochromatin of mammalian cells. Exp Cell Res
60. Hernandez-Verdun D, Gautier T. The chromosome
periphery during mitosis. Bioessays 1994; 16:179-85.
61. Van Hooser AA, Yuh P, Heald R. The perichromosomal
layer. Chromosoma 2005; 114:377-88.
62. Endl E, Gerdes J. The Ki-67 protein: fascinating
forms and an unknown function. Exp Cell Res 2000;
63. Morcos M, Du X, Pfisterer F, Hutter H, Sayed AA,
Thornalley P, et al. Glyoxalase-1 prevents mitochon-
drial protein modification and enhances lifespan in
Caenorhabditis elegans. Aging Cell 2008; 7:260-9.
64. Dettmer J, Hong-Hermesdorf A, Stierhof YD,
Schumacher K. Vacuolar H+-ATPase activity is required
for endocytic and secretory trafficking in Arabidopsis.
Plant Cell 2006; 18:715-30.
65. Bubeck J, Scheuring D, Hummel E, Langhans M,
Viotti C, Foresti O, et al. The syntaxins SYP31 and
SYP81 control ER-Golgi trafficking in the plant secre-
tory pathway. Traffic 2008; 9:1629-52.
66. Friml J, Benkova E, Mayer U, Palme K, Muster G.
Automated whole mount localisation techniques for
plant seedlings. Plant J 2003; 34:115-24.
67. Ritzenthaler C, Nebenfuhr A, Movafeghi A, Stussi-
Garaud C, Behnia L, Pimpl P, et al. Reevaluation of
the effects of brefeldin A on plant cells using tobacco
Bright Yellow 2 cells expressing Golgi-targeted green
fluorescent protein and COPI antisera. Plant Cell
68. Olins AL, Herrmann H, Lichter P, Olins DE. Retinoic
acid differentiation of HL-60 cells promotes cytoskel-
etal polarization. Exp Cell Res 2000; 254:130-42.
23. Foisner R. Cell cycle dynamics of the nuclear envelope.
The Scientific World Journal 2003; 3:20.
24. Haraguchi T, Kojidani T, Koujin T, Shimi T, Osakada
H, Mori C, et al. Live cell imaging and electron micros-
copy reveal dynamic processes of BAF-directed nuclear
envelope assembly. J Cell Sci 2008; 121:2540-54.
25. Fricker M, Hollinshead M, White N, Vaux D.
Interphase nuclei of many mammalian cell types
contain deep, dynamic, tubular membrane-bound
invaginations of the nuclear envelope. J Cell Biol 1997;
26. Broers JL, Machiels BM, van Eys GJ, Kuijpers HJ,
Manders EM, van Driel R, et al. Dynamics of the
nuclear lamina as monitored by GFP-tagged A-type
lamins. J Cell Sci 1999; 112:3463-75.
27. Echevarria W, Leite MF, Guerra MT, Zipfel WR,
Nathanson MH. Regulation of calcium signals in the
nucleus by a nucleoplasmic reticulum. Nat Cell Biol
28. Lui PP, Chan FL, Suen YK, Kwok TT, Kong SK. The
nucleus of HeLa cells contains tubular structures for
Ca2+ signaling with the involvement of mitochondria.
Biochem Biophys Res Commun 2003; 308:826-33.
29. Lee RK, Lui PP, Ngan EK, Lui JC, Suen YK, Chan F,
et al. The nuclear tubular invaginations are dynamic
structures inside the nucleus of HeLa cells. Can J
Physiol Pharmacol 2006; 84:477-86.
30. Olins AL, Buendia B, Herrmann H, Lichter P, Olins
DE. Retinoic acid induction of nuclear envelope-
limited chromatin sheets in HL-60. Exp Cell Res 1998;
31. Melcer S, Gruenbaum Y, Krohne G. Invertebrate lam-
ins. Exp Cell Res 2007; 313:2157-66.
32. Meier I. Composition of the plant nuclear envelope:
theme and variations. J Exp Bot 2007; 58:27-34.
33. Graumann K, Evans DE. The plant nuclear envelope in
focus. Biochem Soc Trans 2010; 38:307-11.
34. Meier I, Brkljacic J. Adding pieces to the puzzling plant
1. Misteli T. Beyond the sequence: cellular organization of
genome function. Cell 2007; 128:787-800.
Kalverda B, Roling MD, Fornerod M. Chromatin
organization in relation to the nuclear periphery. FEBS
Lett 2008; 582:2017-22.
Towbin BD, Meister P, Gasser SM. The nuclear enve-
lope —a scaffold for silencing? Curr Opin Genet Dev
Zhao R, Bodnar MS, Spector DL. Nuclear neighbor-
hoods and gene expression. Curr Opin Genet Dev
Olins DE, Olins AL. Nuclear envelope-limited chro-
matin sheets (ELCS) and heterochromatin higher order
structure. Chromosoma 2009; 118:537-48.
Cremer T, Cremer C. Chromosome territories, nuclear
architecture and gene regulation in mammalian cells.
Nat Rev Genet 2001; 2:292-301.
Cremer T, Cremer M. Chromosome Territories. Cold
Spring Harb Perspect Biol 2010; 2:22.
Lieberman-Aiden E, van Berkum NL, Williams L,
Imakaev M, Ragoczy T, Telling A, et al. Comprehensive
mapping of long-range interactions reveals folding
principles of the human genome. Science 2009;
Guelen L, Pagie L, Brasset E, Meuleman W, Faza MB,
Talhout W, et al. Domain organization of human
chromosomes revealed by mapping of nuclear lamina
interactions. Nature 2008; 453:948-51.
10. de Wit E, van Steensel B. Chromatin domains in higher
eukaryotes: insights from genome-wide mapping stud-
ies. Chromosoma 2009; 118:25-36.
11. Monestier M. Autoantibodies to nucleosomes and
histone-DNA complexes. Methods 1997; 11:36-43.
12. Ballestar E, Esteller M, Richardson BC. The epigenetic
face of systemic lupus erythematosus. J Immunol 2006;
13. Kramers C, Hylkema MN, van Bruggen MC, van
de Lagemaat R, Dijkman HB, Assmann KJ, et al.
Anti-nucleosome antibodies complexed to nucleosomal
antigens show anti-DNA reactivity and bind to rat
glomerular basement membrane in vivo. J Clin Invest
14. Kramers K, Stemmer C, Monestier M, van Bruggen
MC, Rijke-Schilder TP, Hylkema MN, et al. Specificity
of monoclonal anti-nucleosome auto-antibodies
derived from lupus mice. J Autoimmun 1996; 9:723-9.
15. Van Hooser AA, Mancini MA, Allis CD, Sullivan KF,
Brinkley BR. The mammalian centromere: structural
domains and the attenuation of chromatin modeling.
FASEB J 1999; 13:216-20.
16. Moroi Y, Peebles C, Fritzler MJ, Steigerwald J, Tan EM.
Autoantibody to centromere (kinetochore) in sclero-
derma sera. Proc Natl Acad Sci USA 1980; 77:1627-31.
17. Losman MJ, Fasy TM, Novick KE, Monestier M.
Monoclonal autoantibodies to subnucleosomes from a
MRL/Mp(-)+/+ mouse. Oligoclonality of the antibody
response and recognition of a determinant composed
of histones H2A, H2B and DNA. J Immunol 1992;
18. Monestier M, Novick KE. Specificities and genetic
characteristics of nucleosome-reactive antibodies from
autoimmune mice. Mol Immunol 1996; 33:89-99.
19. Hake SB, Garcia BA, Kauer M, Baker SP, Shabanowitz
J, Hunt DF, et al. Serine 31 phosphorylation of histone
variant H3.3 is specific to regions bordering centro-
meres in metaphase chromosomes. Proc Natl Acad Sci
USA 2005; 102:6344-9.
20. McManus KJ, Hendzel MJ. The relationship between
histone H3 phosphorylation and acetylation through-
out the mammalian cell cycle. Biochem Cell Biol 2006;
21. Guttinger S, Laurell E, Kutay U. Orchestrating nuclear
envelope disassembly and reassembly during mitosis.
Nat Rev Mol Cell Biol 2009; 10:178-91.
22. Olins AL, Rhodes G, Welch DBM, Zwerger M, Olins
DE. Lamin B Receptor. Nucleus 2010; 1:1-18.