Novel regulatory regions found downstream of the rat
B29/Ig-
b gene
Ayano Komatsu, Akira Otsuka and Masao Ono
Life Science Course, Department of Chemistry, College of Science, Rikkyo University, Toshima-ku, Tokyo, Japan
To search for novel regulatory regions, we examined the
features of chromatin structure in the rat B29/Ig- b gene and
its flanking regions by determining DNase I hypersensitive
sites (DHS) in plasmacytoma-derived Y3 cells. Six Y3 cell-
specific DHS were detected at )8.6, promoter, +0.7, +4.4,
+6.0, a nd +8.7 kb. The DHS a t + 4.4, +6.0, and +8.7 kb
were present in the intergenic region between B29/Ig- b and
growth hormone (GH) genes and were mapped inside con-
served sequences in rat and humans. In transient transfection
into Y3 cells, 2.9-kb DNA containing the +4.4 and +6.0-kb
DHS demonstrated six times more enhancing activity than
B29/Ig-b promoter alone. Three intergenic DHS each
possessed enhancing activity that was highest in the +4.4-kb
region. In the electrophoretic mobility shift assay, a major
band shift was demonstrated with Y3 nuclear extract and
0.3-kb DNA containing the +4.4-kb region with a con-
served 0.22-kb sequence. By footprint a nalysis, 20 bases in
the middle of the 0.3-kb DNA were protected by Y3 nuclear
extract in which the consensus binding site for the OCT
family was present. Deletion of the footprinted region
reduced enhancing activity to t hat of the B29/Ig-b promoter
alone. The sequence responsible for t he major band shift and
transcriptional enhancing activity in the conserved +4.4-kb
region thus coincided with the 20-bp footprinted region.
Keywords: B29/ Ig-b gene; cell type-specific gene expression;
chromatin structure; conserved regions; D Nase I h yper-

genes are downstream [9]. A mong genes p resent at this
locus, BAF60b and SUG/p45 are house-keeping genes
expressed in all tissues and c ells so far examined, while
SkM sodium channel, B29/Ig-b, GH,andTCAM-1 genes
are expressed cell type-specifically in cells ontogenetically
unrelatedtoeachother.
The B29/Ig- b gene is expressed early in B-cell develop-
ment when Ig genes are still in the g ermline configuration
[10–12]. Mice lacking B 29/Ig-b have completely blocked
B-cell development at the immature B-cell stage [13].
Mouse, human, and rat B29/Ig-b genes lack the TATA
box and have multiple transcriptional initiation sites
[14,15]. In the region starting from the representative
initiation site to 0.17-kb upstream, SP1, ETS, OCT, and
Ikaros motifs essential for B-cell-specific expression of the
mouse B29/Ig-b gene were found [16]. In the 0.35-kb
upstream region of the mouse gen e, two silencer elements
each composed of 30 bp, have been reported [17]. In the
0.5-kb upstream region of the human gene, a 30-bp
positive transcription control element is present [16]. An
early B-cell factor (EBF) essential for early B lymphocyte
development has been shown to be involved in B29/Ig-b
gene expression from the promoter region to 0.17-kb
upstream [18]. Thus, the me chanism of B -cell-specific
Correspondence to M. Ono, Life Science Course, Department of
Chemistry, College of Science, Rikkyo University, 3-34-1 Nishi-
ikebukuro, Toshima-ku, Tokyo 171-8501 Japan.
Fax/Tel.: + 81 339852387, E-mail: [email protected]
Abbreviations:BCR,B-cellreceptor;DHS,DNaseIhypersensitive
sites; D-MEM, Dulbecco’s modified minimal essential medium; EBF,

specific DHS in the B29/Ig-b gene and its flanking regions
were examined in rat Y3 cells expressing B29/Ig-b mRNA.
In the region between th e B29/Ig-b and GH genes, three Y3
cell-specific DHS were found and each possessed transcrip-
tional enhancing activity for transie nt transfection into Y3
cells. The nucleotide sequenc es of these DHS are conserved
in rat and humans. Of these DHS, the 0 .3-kb region with the
highest enhancing activity was analyzed exclusively.
MATERIALS AND METHODS
Cells, clones, Northern hybridization, and sequencing
Rat plasmacytoma-derived Y3-Ag1.2.3 cells were obtained
from the Japanese Cancer Research Resources Bank
(Tokyo, Japan). Buffalo rat liver-derived BRL cells were
obtained from RIKEN Cell Bank (Tsuku ba, Japan). Rat
pituitary-derived GC cells were obtained from M. Karin
(University of California, San Diego, CA, USA). The Y3
and BRL cells were propagated in Dulbecco’s modified
minimal essential medium (D-MEM)/10% fetal bovine
serum ( JRH Bioscience s, US A) and the GC cells were
Fig. 1. Expression of B29/Ig-b mRNA (A) and DNase I hypersensitivity by SkM sodium channel and B29/Ig-b gene chromatins of Y3 cells (B).
(A) Poly ( A)-rich RNA (3.0 lg) electrophoresed on a 2.2-
M
HCHO/1% agarose gel and hybridized with
32
P-labeled rat cDNA (nucleotides 74 to
880) [3]. The RNA size (given in kb) was determined using a commercial RNA molecular size marker (Roche Diagnostics). At the bottom,
reprobing with GraP-DH is shown. (B) Isolated Y3 nuclei t reated with DNase I for 3 min at 20 °C. Concentration of DNase I for treatment of
nuclei from left to right: 200, 100, 50, 25, 13, 0 UÆmL
)1
. T he D NA was purifi ed from nuclei and digested with NheI. The digests (2.0 lg) were

hybridization
The Y3 nuclei were prepared in N1 buffer (15 m
M
Tris/HCl,
pH 7.5, 60 m
M
KCl, 15 m
M
NaCl, 5 m
M
MgCl
2
,0.5m
M
dithiothreitol, 0.1 m
M
EGTA, 0.3
M
sucrose) containing
0.2% NP-40 as described previously [24]. For DNase I
(TaKaRa Shuzo, Kyoto, Japan) digestion, 60 lLofenzyme
in N1 buffer was mixed i n 750 lLwith3·10
7
nuclei and
incubated for 3 min at 20 °C. Reactions were terminated by
the addition of 75 lLstopsolutioncontaining5%SDSand
100 m
M
EDTA. The DNA preparation, restriction enzyme
digestion, and Southern hybridization were carried out as

Pyrobest polymerase (TaKaRa, Kyoto, Japan) amplifica-
tion of th e B29/Ig-b promoter (nucleotides )201 to +30)
Fig. 2. DNase I hypersensitivity of SkM sodium channel and B29/Ig-b gene chromatins. DNase I treatment and Southern hybridization were carried
out as previously described. The DNA was digested with ApaI(A)orHindIII (B) and hybridized with a 0.6-kb probe (nucleotides 1571 to 2158).
Left, Y3 ; right, BRL.
Ó FEBS 2002 Rat B29/Ig-b gene regulatory regions (Eur. J. Biochem. 269) 1229
[3] followed by cloning into the HindIII/BglII sites of
PGV-B2. The B29/Ig-b promoter sequence was selected
for its similarity of those of mouse and humans [15,16].
The DHS were amplified with a cosmid template and
cloned into the MluI/NheI sites of B29-P or SV-P. For
the )8.6-kb unsequenced DHS region, 0.8-kb DNA was
obtained by generating XbaI()8.9 k b)/HindIII ()8.1 k b)
digestion of c osmid DNA. Activity in t he following
regions (nucleotide numbers from transcriptional start site
of B29/Ig-b gene) was examined by transfection: +0.7 kb
(540–809), +4.4a (4193–4483), +4.6b (4451–4727), +6.0c
(5902–6227), +6.5d (6352–6631), +4.4a to +4.6b (4193–
4727), + 6.0c to +6.5d (5902–6631), +8.7 kb ( 8630–9238),
and +3.8 kb to + 6.7 k b (3831–6652). The activities of
at least two i ndependent clones were determined to b e
the same, so the results of a r epresentative clone are
shown.
A reporter plasmid lacking a 30-bp sequence (4313–
4342) including the 20-bp footprinted region was produced
as follows: two 21-bp primers (5¢ 4343–4363 3¢;5¢4312–
4292 3¢)withaSpeI site and three more nucleotides a t the
5¢ end were used for amplification by Pyrobest polymerase
with the B29-P reporter t hat h ad the +4.4a DHS.
Amplified DNA was digested with SpeI, ligated, and

M
EDTA, 1.5 m
M
MgCl
2
,1m
M
dithiothreitol, 1% protease inhibitor cocktail, 20% g ly-
cerol), then centrifuged again at 25 000 g for 30 min at
4 °C. The supernatant was used for electrophoretic mobility
shift and footprint assays [25]. A BCA protein assay kit
(Pierce, USA) was used for p rotein determination. For end-
labeling, +4.4a DNA (4193–4483) was digested with MluI
and then labeled with Klenow enzyme (TaKaRa), dGTP,
and [a-
32
P] dCTP. The binding reaction was first carried out
without labeled DNA in 17 lL binding buffer (10 m
M
Hepes/KOH pH 7.8, 40 m
M
KCl, 1 m
M
EDTA, 5 m
M
MgCl
2
,0.12mgÆmL
)1
poly(dI–dC) (Amersham Pharmacia

M
EDTA, 50 lgÆmL
)1
salmon
sperm DNA). The DNA was extracted with phenol,
precipitated with ethan ol, and analyze d on a 5% polyacryl-
amide sequencing gel.
RESULTS
Y3 cell-specific DNase I hypersensitive sites
B29/Ig-b mRNA was detected in rat plasmacytoma-derived
Y3 cells by Northern hybridization with a rat B29/Ig-b
cDNA probe (Fig. 1A). The features of the chromatin
structure in the B29/Ig-b gene and its flanking 15-kb
upstream and 31-kb downstream regions were then exam-
ined by locating the DHS. In the upstream region, DHS at
the promoter and at )11.2 kb and )8.6 kb inside the
sodium channel g ene were fo und (Fig. 1B). Another site at
+0.7 kb was located between exons 1 and 2 in the B29/Ig-b
gene, four sites (+4.4, +6.0, +8.7, +11.2) were present
between the B29/Ig-b and GH genes (Fig. 2), and two sites
were found at +15.1 kb and +20.8 kb, between the GH
and TCAM-1 genes (data not shown). I n t he B29/Ig- b
nonproducing B RL and the GH-pro ducing GC cells, DHS
at )11.2 kb and +11.2 kb were observed [7]. Sites at
+15.1 kb and +20.8 kb are also present in GC cells [7]. Six
DHS ()8.6, promoter, +0.7, +4.4, +6.0, +8.7) were
found to be Y3 cell-specific.
Relation of Y3 cell-specific DHS to conserved regions
Regions including cell type-specific DHS have been found
to correspond not only to promoters and enhancers, but

transcriptional enhancing activity [19,20] and t his activity
was sought for the present s tudy (Fig. 4). The Y3 cell-
specific DHS in and around the B29/Ig-b gene were inserted
upstream in the B29/Ig-b promoter plasmid (B29-P)with
the firefly luciferase reporter gene. Recombinant plasmids
were transiently transfected into Y3 cells and the luciferase
activity was measured. The reporter plasmid with the B29/
Ig-b promoter ()201 to +30) possessed 21 times more
luciferase activity than the pro moterless control. No signifi-
cant activity was found in the 0.8-kb DNA prepared from
the Y3 cell-specific )8.6-kb site or the 0.27-kb fragment
from the +0.7-kb region. The 2.9-kb DNA from +3.8 kb
to +6.7 kb containing the +4.4a to +6.5d regions showed
six t imes more enhancing activity than the B29/Ig-b
promoter alone. In the +4.4a to +6.5d regions, the highly
conserved +4.4a region with one of the Y3 cell-specific
DHS s howed the greatest luciferase a ctivity; th e +6.0c
region showed the second greatest. Two less conserved but
non-DNase I hypersensitive regions, +4.6b and + 6.5d,
had n o a ctivity. A 1.1-kb fragment from the +8.7-kb region
was two times more active than the B29/Ig-b promoter,
although its enhancing activity was less than that for the
+6.0c region alone.
Experiments were c on ducted to determine whether
regions containing transcriptional enhancing activity are
orientation- or promoter-dependent (Fig. 5). Reverse-
orientation 2.9-kb DNA containing the +4.4a to +6.5d
regions displayed even more enhancing activity than
normally oriented DNA. Reverse-orientation +4.4a DNA
was 2.5 time s more active than the B29/Ig-b pro moter alone

shift is present in this region.
Fig. 6. Nucleotide sequence comparison of the
conserved + 4.4a region in rat and humans.
Upper, rat; lo wer, humans. Nucleotide
numbers from transcriptional start site o f
the B29/Ig-b gene are indicated. Identical
nucleotides are shown by asterisks (*). The
conserved region i s enclosed with lines.
Potential binding sites o f the transcription
factors are boxed.
1232 A. Komatsu et al. (Eur. J. Biochem. 269) Ó FEBS 2002
To further deline ate the major binding site, f ootprint
analysis was carried out. Each 3¢ end of the 0.3-kb DNA
with the + 4.4a region was labeled with
32
P for footprinting
(Fig. 8). Nucleotides 4320 to 4339 and 4337 to 4320 from
the 5¢ end of the B29/Ig- b gene were protected in forward
and reverse strands (Fig. 8). In the protected regions, a
sequence was found corresponding to the binding site for
the OCT family transcription factor [29]. Presumed S RY
binding sites GATA and NF-kB were present in the
conserved +4.4a region (Fig. 6) but they were not pro-
tected. To determine whether the footprinted region is
responsible for the major electrophoretic mobility shift
activity, a 30-bp fragment from nucleotides 4313 t o 4342
that include the p rotected region was u sed for competition
(Fig. 7). This fragment outcompeted the major band,
whereas the fragment from 4403 to 4432 did not, suggesting
that the footprinted region is r esponsible for the major

as far as the 1.2-kb upstream region and their interac-
ting transcription f actors. During B -cell development,
B-cell-specific gene expression seems to require not only
Fig. 7. Binding of Y3 nuclear proteins to +4.4a DNA.
32
P-Labeled
0.3-kb DNA containing the +4.4a region was incubated for 3 0 m in at
room temperature with Y3 nuclear extract (0.7 lg). Competition
reactions were performed using full 0.3-kb DNA, a HinfI fragment
(nucleotides 4193–4397), footprinted fragment A (nucleotides 4313–
4397), and control fragment B (nucleotides 4403–4432). The amount of
unlabeled competitor is indicated as fold mo lar excess. Nucleotide
numbers from transcriptional start site of the B29/Ig-b gene are shown.
Fig. 8. DNase I footprint analysis of the +4.4a region. The 0.3-kb
DNA (nucleotides 4193–4483) containing the +4.4a region labeled
with
32
P was u sed. DNA binding reactions were incubated for 30 min
at 25 °C with Y3 nuclear extract. F, coding strand; R, noncoding
strand. Footprinted regions are indicated in margins. At bottom,
features of the 0.3-kb DNA and nucleotide sequence of the footprinted
region are s hown. N ucleotides 4245–4461 are the conserved region.
Nucleotide numbers from transcriptional start s ite of the B29/Ig-b
gene are indicated.
Ó FEBS 2002 Rat B29/Ig-b gene regulatory regions (Eur. J. Biochem. 269) 1233
recruitment of transcription factors and adaptors pre-
requisite for gene expression, but also change in the
chromatin structure from an inactive st ate t o an active
state. Cis-elements for transcription factors or the regions
required f or the structural change of the chromatin are often

Three intergenic DHS corresponding +4.4a, +6.0c, and
+8.7e were each found to possess enhancing a ctivity when
inserted into the B29 promoter reporter and transfected into
Y3 cells (Fig. 4). Because 2.9-kb DNA containing +4.4a to
+6.5d regions exhibited higher enhancing activity than e ach
of the three regions individually, the +4.4a to +6.5d
regions along with +8.7e s hould be involved in B29/Ig-b
gene expression. Neither the 2.9-kb DNA nor any of the
three regions individually had any enhancing/silencing
activity when combined with the GH promoter reporter
and transfected into GH-producing G C cells (data n ot
shown) and thus are regulatory regions for B29/Ig-b gene
expression. The LCR often coincides with a cluster of cell
type-specific DHS and some DHS in the L CR po ssess
transcriptional enhancing a ctivity [21,22]. Typical LCRs
such as human b-glo bin, human CD2, and mouse k5/
VpreB1 loci have these features [21]. Four cell type-specific
DHS that comprise the human b-globin LCR are present in
the i ntergenic region between an odorant receptor gene
cluster and the b-globin locus [33] and their nucleotide
sequences are conserved in mouse and humans [27]. The
features of three intergenic DHS demonstrated in the
present study correspond well to those of the LCR, so these
DHS likely form the LCR of the B29/Ig-b gene, although
further examination using transgenic or knockout mice
should be conducted to confirm this point.
Mammalian g enes are often separated by long intergenic
regions in which regulatory regions for transcription are
scattered, thus making it difficult to find them. As shown this
and previous studies [26,28], novel transcriptional r egulatory

ment of H3 and H4 histone acetylation was observed from
the promoter region of human GH gene to the upstream
region present in the sodium channel gene in the chromatin
of GH-producing cells [39]. In B29/Ig-b-producing cells,
whether enhancement of core h istone acetylation along with
Fig. 9. Enhancing activity o f deletion constructs of the f ootprinted
region. The 30-b p sequ en ce includ in g t he footprin ted r egion (nucleo-
tides 4313–4342) deleted f rom the reporter construct having both the
+4.4a region (nucleotides 4193–4474) and B29-P. Nucleotide numbers
from transcriptional start site of the B29/Ig-b gene are shown. Deletion
from nucleotides 4193–4222 was used for comparison.
1234 A. Komatsu et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the general sensitivity to DNase I is present not only in th e
B29/Ig-b gene itself but also in flanking intergenic regions
and the sodium chan nel and GH genes is a point of interest.
ACKNOWLEDGEMENTS
We th ank M. Karin, the Japanese Cancer Research Resources Bank,
and RIK EN Cell Bank for p roviding the cells. This w ork w as
supported by a grant from the Foundation of Growth Sciences and b y
Rikkyo University f or the Promotion of Research.
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