The EMBO Journal vol.10 no.5 pp.1225- 1236, 1991
The maternally expressed Drosophila gene encoding the
chromatin-binding protein BJ1 is a homolog of the
vertebrate gene Regulator of Chromatin Condensation,
Max-Planck-Institut fur Entwicklungsbiologie, Abt.
Communicated by C.Nusslein-Volhard
1 and 3, D-7400
Using monoclonal antibodies I have identified a nuclear
protein ofDrosophila, BJ1 (Mr
gene. Biochemical analysis demonstrates that the BJ1
protein is associated with nucleosomes and is released
from chromatin by agents which intercalate into DNA,
as previously shown for the high mobility group proteins
(HMGs). On polytene chromosomes the protein
localized in all bands, with no preference for particular
loci. Both the BJ1 protein and in particular the BJ1
mRNA are strongly expressed maternally. In early
embryos all nuclei contain equal amounts of BJ1. During
neuroblast formation, BJ1 mRNA becomes restricted to
cells of the central nervous system, and higher protein
levels are found in the nuclei of this tissue. In late
the mRNA almost completely
disappears, but significant amounts ofBJ1 protein persist
until morphogenesis. The BJ1 gene encodes a 547 amino
acid polypeptide featuring two different types of internal
repeats. The sequence from amino acids 46 to 417
containing seven repeats ofthe firt type has been highly
conserved in evolution. 45% of the amino acids in this
region are conserved in seven si'milar tandem repeats of
the human gene Regulator of Chromatin Condensation,
RCCI. The phenotype of a cell line carrying a mutation
of RCCI suggested a main function for this gene in cell
cycle control. A yeast gene, SRMI/PRP20, also contains
these repeats and shows 30% amino acid identity to BJ1
in this region. Mutations in this gene perturb mRNA
metabolism, disrupt nuclear structure and alter the signal
for the mating pheromone.
Complementation experiments argue for a common
function ofthese genes in the different species. I propose
that their gene products bind to the chromatin to establish
or maintain a proper higher order structure as a
prerequisite for a regulated gene expression. Disruption
of this structure could cause both mis-expression and
default repression of genes, which might explain the
pleiotropic phenotypes of the mutants.
Key words: cell cycle control/chromatin structure/non-
68 kd), and isolated its
In nuclei of eukaryotic cells the DNA is complexed with
proteins to form a compact structure, the chromatin. The
basic unit of the chromatin is the nucleosome, which contains
an octameric core of four different histones with
ofDNA wrapped around them. The nucleosomes can form
Oxford University Press
higher order structures, resulting in variable degrees of
chromatin condensation along the interphase chromosomes.
It appears that not only the presence of specific transcription
factors (activators or repressors), but also the degree of
chromatin condensation may play a decisive role in the
expression or repression of a gene (Weinstraub and
Groudine, 1976; Wu et al., 1979; reviewed by Felsenfeld
and McGhee, 1986; Widom, 1989). Thus, inactivation of
genes can occur when these are brought into the vicinity of
rearrangements (reviewed by Henikoff, 1990). In normal
development particular states of expression can be stably
maintained even after the disappearance of regulatory
proteins that are required to establish expression or
repression initially. It has been proposed that this imprinting
ofgenetic activity occurs via the chromatin structure (Paro,
Many data have accumulated pointing to the role of both
the histones and the non-histone proteins in determining the
higher order chromatin structure of genomic regions. In
particular, the presence of histones HI, Hio, or H5, which
bind to the linker regions between the nucleosomes, can
induce assembly ofmore condensed structures and repression
of genes (Schlissel and Brown, 1984; Sun et al., 1989;
Wolffe, 1989). Of the non-histone proteins influencing
chromatin structure, the class of the moderately abundant
high mobility group proteins (HMGs) has been studied in
most detail. The term HMG proteins has been operationally
defined according to biochemical properties that facilitate
isolation of these proteins (reviewed in Johns, 1983). While
HMG14 and HMG17 are preferentially associated with
decondensed, transcriptionally active chromatin (Weisbrod
et al., 1980; Dorbic and Wittig, 1987), other HMGs such
as the a-protein are thought to induce specific positioning
of nucleosomes and higher order chromatin structures
(Strauss and Varshavsky, 1984). However, the function of
these proteins is far from being clear (Einck and Bustin,
1985). Presumably there exist additional proteins influencing
chromatin structure which have not been isolated because
of their lower abundance or because their biochemical
properties differ from those of the HMGs.
Monoclonal antibodies have been successfully used to
dissect the complexity of nuclear protein fractions and to
analyze the functions of their individual components
(Saumweber et al., 1980. Dreyer et al., 1981; Kuo et al.,
1982; Lacroix et al., 1985; Garzino et al., 1987). In
Drosophila, this approach has allowed the identification of
nuclear proteins involved in gene expression (Risau et al.,
Frasch and Saumweber,
condensation (James and Elgin, 1986; Eissenberg etal.,
1990), and in structural functions of the nucleus (Fuchs
et al., 1983; Frasch et al ., 1988). In such an immunological
screen, I obtained monoclonal antibodies against39 different
nuclear proteins (Frasch,
obtained class of antibodies recognizeda 68 kdantigenwith
1985). The most frequently
moderate abundance in embryonic nuclei, called BJ1. Here,
I describe the chromatin binding of BJ1, its expression
pattern, and the cloning and sequencing of the BJ1 gene.
The sequence of BJ1 turned out to be strongly conserved
in evolution, and genes coding for related proteins are found
from yeast to humans. A homologous gene in vertebrates,
Regulator ofChromatin Condensation (RCCJ), was studied
most extensively, and its main function appears to be in cell
cycle control (reviewed in Nishimoto, 1988). In a hamster
cell line with a temperature sensitive mutation for RCCJ,
premature initiation of mitosis occurs when the cells are
shifted to the restrictive temperature during S phase (Ajiro
et al., 1983; H.Nishitani, M.Ohtsubo, K.Yamashita, H.Iida,
J.Pines, H.Yasuda, Y.Shibata, T.Hunter and T.Nishimoto,
submitted). However, temperature shifts inGIphase result
in a different phenotype, and cause alterations in gene
expression (Nishimoto et al., 1981). In yeast, the phenotypes
ofmutations in a homologous gene, called SRMI (Clark and
Sprague, 1989) or PRP20 (Aebi et al., 1990), are also com-
patible with the idea that the gene product is required for
normal gene expression and chromatin structure. The pro-
perties of the Drosophila BJ1 protein provide important clues
to the function(s) of these proteins in the nucleus and may
explain the pleiotropic phenotypes observed in these mutants.
Monoclonal antibodies against BJ 1
Monoclonal antibodies were produced against protein
fractions from Drosophila embryonic nuclei. Forty one
independent hybridoma clones obtained from one particular
protein fraction (see Materials and methods) secreted anti-
bodies against nuclear proteins. On Western blots, 29 of
these antibodies recognized an antigen with an electrophoretic
mobility of 68 kd present in total nuclear proteins from
embryos (Figure IA, lane 3) and from Drosophila tissue
culture cells (KC cells, Figure IA, lanes 4-7). Several lines
ofevidence suggested that the antibodies recognized at least
four different epitopes in this 68 kd polypeptide, which I
call BJ1. The four classes of antibodies were represented
by the antibodies BjlO, Bj43, Bj59, and Bj7O. These
antibodies reacted with different peptides after partial
digestion ofBJ1 with V8 protease (data not shown; Cleveland
et al., 1977). BJ1/3Gal fusion proteins of different length
allowed the partial mapping of the binding regions of these
antibodies within the BJ1 polypeptide. The antibodies also
differed in terms of their crossreaction with proteins from
other species (see below). Furthermore, with nuclear proteins
from Kc cells they recognized minor bands in addition to
the 68 kd band in four different patterns (Figure IA).
Interestingly, the 60 kd band recognized by the Bj7O
antibody (Figure lA, lane 7) appears to be identical to the
nuclear protein DI described previously (Alfageme et al.,
1980; Levinger and Varshavsky, 1982a,b). The Dl protein
is quantitatively extracted from nuclei with 5% perchloric
acid (PCA), together with only few other proteins including
histone Hi and A13 (Bassuk and Mayfield, 1982. Figure
iB, lanes 2 and 4). In a Western blot with the Bj7O antibody,
a 60 kd band corresponding to DI was stained with the PCA
extract, whereas BJ1 (68 kd) remained in the insoluble
fraction (Figure 1B, lanes 6 and 7). Thus, the nuclear
proteins BJ1 and D1 ofDrosophila are antigenically related.
Two classes of Bj antibodies also crossreacted with
proteins from species that are very distantly related to
2 3 4 5 6
Fig. 1. Characterization of BJ1 and related proteins by Western
blotting. A. Western analysis of Drosophila nuclear proteins with four
different antibodies against BJ1. Lanes 1 and 2:total nuclear proteins
from embyros or KC cells, respectively, stained with Coomassie
brilliant blue. Lane 3: Western blot of embryonic nuclear proteins, as
in lane 1, with Bj43 antibody. Lanes 4-6: Western blot of Kc cell
nuclear proteins, as in lane 2, with the antibodies Bj43 (lane 4), BjlO
(lane 5), Bj59 (lane 6) and Bj7O (lane 7) B. Extraction and Western
analysis of DI protein. Lanes 1-4: Coomassie stainings of total
nuclear proteins form KCcells (lane 1), 5% perchloric acid extract
enriched for Dl (lane 2 with 3Atgof protein loaded, lane 4 with
30Ag),and residual nuclear proteins after PCA extraction (lane 3).
Lanes 5-6: Western analysis of immobilized proteins as in lanes 1-3
with Bj7O antibody. C. Cross reaction of BJ1 antibodies with proteins
from nuclei of embryonic chicken (lanes 1 and 4), with proteins from
calf liver nuclei (lanes 2 and 5) and with proteins from total yeast cells
(lanes 3 and 6). Lanes 1-3 were stained with Bj59 antibody, lanes
4-6 with Bj7O antibody.
Drosophila. On Western blots with nuclear proteins from
embryonic chicken brains and calf liver, bands of55-64 kd
were stained with the Bj59 class, and two bands of-80 kd
and 90 kd with the Bj7O antibody class (Figure IC, lanes
1, 2, 4 and 5). These antibodies exclusively stained the nuclei
on tissue sections from chicken embryos (data not shown).
On Western blots with total proteins from yeast, two bands
were recognized by the Bj59 antibody class (69 kd and
84 kd), and a weak band by the Bj7O antibody class (40 kd).
These results suggest that the epitopes recognized by these
antibodies have been conserved in several nuclear proteins
Chromatin-binding of BJ1
Since BJ1 was found to be antigenically related to the HMG-
like protein Dl, I examined if it shared any biochemical
properties with HMG proteins. In particular, I tested whether
BJ1 (and Dl) is specificallv released from nuclei upon
alteration ofthe DNA conformation by intercalation. 'Elutive
intercalation' has been used previously to release HMG-like
proteins and other DNA-binding proteins from whole nuclei.
The composition of the eluted protein fraction depended on
the nature and concentration of the intercalating agent used,
and on the ionic strength (Schroter et al., 1985, 1987;
Schulman et al., 1987).
When nuclei from Kc cells were treated with 7.5 mM
ethidium bromide and 50 mM NaCl, the eluted protein
contained one major band with a mobility of 68 kd (Figure
2A, lane 3). This band corresponded to the BJl protein, as
shown by western analysis (Figure 2B, lane 3) and by co-
purification of the antigenic activity with the 68 kd band,
when the extract was further fractionated on an FPLC
MonoS column (data not shown). At a slightly higher ionic
100 mM NaCl, BJI was eluted even more
Expression, localization and chromatin-binding of BJ1 protein
Drosophila and yeast should help to further clarify the rela-
tionship between chromatin structure, gene expression and
cell cycle control.
Materials and methods
Isolation of nuclei
10 g portions of 1- 15 h old embryos were dechorionated and homogenized
in 100 ml ofembryo buffer (60 mM KCI, 15 mM NaCl, 0.34 M sucrose,
1 mM DTE, 1 mM EDTA,
X-100, 15 mM Tris pH 6.4). The suspension was filtered through a nylon
mesh and layered onto step gradients of 3 ml 2.4 M sucrose and 7 ml 1.3 M
sucrose in embryo buffer. After centrifugation for 15 min at 6000 r.p.m.
in a Sorvall HB4 rotor, the pellets on top of the 2.4 M sucrose cushion
were homogenized and diluted in 150 ml of embryo buffer. The nuclei were
recentriftiged for 90 min at 25000 r.p.m. in a SW27 rotor on step gradients
of 1 ml 2.4 M sucrose and 10 ml 2.2 M sucrose in embryo buffer. The
nuclei were collected from the top phase of the 2.4 M sucrose cushion.
Nuclei from Kc cells were isolated as described previously (Risau et al.,
1 mM EGTA,
1 mM PMSF, 0.1 % Triton
Generation of monoclonal antibodies against nuclear proteins
The preparation of fractions of nuclear proteins used for immunization has
been described (Frasch and Saumweber, 1989). The Bj antibodies were
obtained with a fraction of proteins that were released from chromatin at
450 mM NaCl and that were contained in the flow through of a QAE
Sephadex column at 5 M urea, 150 mM NaCl, 0.5 mM DTE, 10 mM Tris
pH 8.3 (Augenlicht and Baserga, 1973). BjlO, Bj46, Bj59 and Bj70 are
IgG1, Bj43 is IgG3, and Bj86 isIgG2h.
Preparation and fractionation of soluble chromatin
The digestion of nuclei fromKccells with micrococcal nuclease and RNase
A, the fractionation of the extracted chromatin on sucrose gradients and
the analysis of the gradient fractions were done as described previously
(Frasch and Saumweber, 1989).
Protein extraction by 'elutive intercalation'
This procedure was performed according to Schroter et al.
4.5 x 108 nuclei from Kc cells were resuspended in 3 ml of X2 buffer
(1 mM Tris pH 7.0, 0.2 mM EDTA), 7.5 mM ethidium bromide, and NaCl
at concentrations of 0, 50 or 100 mM. Extraction was for 45 min at 0°C
with shaking. After centrifugation (10 min, 4000 g), the extracted proteins
were precipitated with 20% TCA, washed with ethanol, and boiled in SDS
gel loading buffer. The extract recovered from 5 x 107 nuclei (Coomassie
staining) or 1 x 107 nuclei (Western blots) was loaded in each lane.
Gel electrophoresis and Western blots
Electrophoresis on SDS-polyacrylamide gels and Western blots were done
as described (Frasch and Saumweber, 1989). The following proteins were
used as molecular weight standards: trypsin inhibitor (soybean, 20.1 kd),
carbonic anhydrase (29 kd), P11 antigen (Risau et al., 1983; 36 kd), alcohol
dehydrogenase (subunit, 39.8 kd), BSA (68 kd), S5 antigen (Risau et al.,
1983; 70 kd), ,B-galactosidase (E.coli, 116 kd), RNA polymerase (E.coli,
155 kd and 165 kd) and myosin (205 kd).
Isolation of cDNA clones
cDNA clones were isolated from a Xgtl 1 expression library as described
by Young and Davis (1983). The library was made from 0-2 h embryonic
RNA with a size-selection
(U.Rosenberg, unpublished). 240 000 phages were plated with a density
of 14 000 phages per plate (11 x 11 cm). After IPTG induction, a mixture
of the monoclonal antibodies Bj43, Bj46, BjS9 and Bj7O, binding to different
epitopes of the BJl protein, was used for filter incubation, followed by a
secondary, alkaline phosphatase conjugated antibody. After the staining
reaction, signals were obtained from 23 phages, 12 of which were further
purified. Three of these could be detected by all four antibodies, two were
only detected by Bj46, four were only detected by BjS9, and nine of them
showed cross-hybridization of their inserts.
cDNAs with longer inserts were isolated from a pNB40-plasmid library
prepared from 4-8 h embryonic RNA (Brown and Kafatos, 1988).
in the 500 bp range
cDNAs and genomic fragments were subcloned into pBluescript KS + and
SK+ (Stratagene). Deletions were created with appropriate restriction
enzymes and the sequencing reactions were carried out with M13 primers
or with primers complementary to insert sequences. Sequencing was
performed by the dideoxynucleotide sequencing method (Sanger et al., 1977)
using single stranded templates and a modified form of T7 polymerase
Northern blots were done as described previously (Dohrmann et al., 1990),
but using total RNA rather than poly(A)+ RNA.
In situ hybridization
In situ hybridizations to whole embryos and ovaries were carried out ac-
cording to the procedure ofTautz and Pfeifle (1989). Digoxigenin conjugated
dUTP and anti-digoxygenin antibodies were from Boehringer Mannheim.
Hybridization probes were prepared according to the protocol of Feinberg
and Vogelstein (1984), using a molar ratio ofdTTP:DigdUTP of 2:1. For
sections (10/Am),the embryos were embedded in Araldite after staining
(Leptin and Grunewald, 1990).
For antibody stainings ofwhole embryos (Dequin et al., 1984) and ovaries,
fixation was carried out for 20 min in a buffer containing 45 mM KCI,
15 mM NaCl,
13 mM MgCl2,10 mM K-phosphate pH 6.8, 4%
formaldehyde and 15% (v/v) of a saturated, aqueous solution ofpicric acid.
When picric acid was not included, the cytoplasmic BJI protein was not
retained. Frozen sections were made after the staining (Dequin et al., 1984).
Hoechst 33258 was used for DNA staining at 1 Stg/ml (in PBS). For HRP
stainings, the VECTASTAIN detection kit (Vector Laboratories) was used
with diaminobenzidine as a substrate.
Fixation, squashing and staining of polytene chromosomes was done as
described by Saumweber et al. (1980). Photographs were taken with Kodak
2415 technical pan film and developed with developer HCl 10 (Nomarski
optics) or D19 (fluorescence).
I gratefully acknowledge the contributions of Raymond Dequin at the
beginning of the project and would like to thank Harry Saumweber for his
advice. I appreciate the expert technical assistance ofMonika Wild and Meike
Muller. I would like to thank Friedrich Bonhoeffer and Janni Niisslein-
Volhard for their support and encouragement. I also thank Takeharu
Nishimoto for communicating unpublished results, and Christian Lehner
and Daniel St Johnston for critically reading the manuscript.
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Received on January 30, 1991