Tài liệu Báo cáo khoa học: Effect of deletion of the DNase I hypersensitive sites on the transcription of chicken Ig-b gene and on the maintenance of active chromatin state in the Ig-b locus docx

Effect of deletion of the DNase I hypersensitive sites on
the transcription of chicken Ig-b gene and on the
maintenance of active chromatin state in the Ig-b locus
Hiroki Matsudo
1
, Kyoichi Osano
1
, Hiroshi Arakawa
2
and Masao Ono
1
1 Department of Life Science, and Frontier Project ‘Life’s Adaptation Strategies to Environmental Changes’, Rikkyo University,
College of Science, Toshima-ku, Tokyo, Japan
2 GSF, Institute for Molecular Radiobiology, Neuherberg-Munich, Germany
In vertebrate cells, chromatin of active or potentially
active genes and flanking regions are characterized by
(a) sensitivity to DNase I [1–6]; (b) the presence of cell
type-specific DNase I hypersensitive sites (DHSs) [7,8];
and (c) core histone modifications such as acetylation
and methylation specific for active chromatin [9–11].
These characteristics have been reported specifically
for loci such as b-globin [1,3,12,13], and Ig-b ⁄ growth
hormone (GH) [14–16]. Thus, it is possible to differen-
tiate between active, or potentially active, and inactive
chromatin states by comparing differences in chroma-
tin sensitivity to nuclease and histone modifications.
However, the mechanism by which the chromatin
structure is modified in order to initiate cell type-speci-
fic gene expression as well as the maintenance of this
active state in differentiated cells remains to be elucida-
ted [17–19]. Along with membrane immunoglobulin

and Ig-a ⁄ mb1, Ig-b is a component of the antigen
receptor complex and belongs to the immunoglobulin
superfamily [20,21]. The Ig-b gene is expressed early in
B cell development [22–24]. The mechanism of B cell-
specific expression of mouse and human Ig-b genes has
been studied mainly in the proximal promoter region,
and several cis-elements and transacting factors have
Keywords
chicken Ig-b gene; DNase I hypersensitive
sites; DT40; histone acetylation;
transcription
Correspondence
M. Ono, Department of Life Science,
College of Science, Rikkyo University,
Toshima-ku, Tokyo 171-8501, Japan
Fax ⁄ Tel: +81 339852387
E-mail:
(Received 11 September 2004, revised 3
November 2004, accepted 15 November
2004)
doi:10.1111/j.1742-4658.2004.04482.x
The role of DNase I hypersensitive sites (DHSs) in transcription of the B
cell-specific Ig-b gene and in maintenance of active chromatin state in the
Ig-b locus were examined. A total of 10 DHSs were divided into four
regions, and each region was deleted separately in chicken B lymphocyte-
derived DT40 cells. Deletion of three DHSs located between the Ig-b pro-
moter and its upstream Na channel gene, resulted in the absence of Ig-b
mRNA. Three regions except the region in the Na channel gene were
involved in the transcription of Ig-b gene. The enhancing activity of DHSs
as determined by transient transfection assays did not always correlate with

the effect of DHS deletion on the expression level of Ig-b mRNA. In each
deletion, cells contained the same DHSs as observed in the predeletion
cells, indicating that deleted DHSs did not participate in the maintenance
of DT40-specific DHSs. Enhanced acetylation of H3 and H4 histones at
the Ig-b promoter and at DT40-specific DHSs was observed in cells in
which DHSs between the Na channel gene and Ig-b promoter were deleted;
therefore, these DHSs are prerequisite for transcription of the Ig-b gene
but not required for the maintenance of active chromatin state in the Ig-b
locus. Thus, epigenetic factors required for the maintenance of the active
chromatin state are suggested to reside in other regions than those deleted
in this study.
Abbreviations
ChIP, chromatin immunoprecipitation; DHS, DNase I hypersensitive site; GH, growth hormone; LCR, locus control region; R-PCR, real-time
polymerase chain reaction.
422 FEBS Journal 272 (2005) 422–432 ª 2004 FEBS
been identified [25–27]. However, these studies have
not taken the state of chromatin into consideration.
Cell type-specific DHSs not only correspond to pro-
moters and enhancers but also are likely to participate
in the establishment and maintenance of an active
chromatin state [18,28,29]. In addition to their pres-
ence in and adjacent to active or potentially active
genes, cell type-specific DHSs are found in regions
situated far upstream or downstream of a gene [1,7,8].
To identify novel regions participating in cell type-spe-
cific transcription of the rat Ig-b gene, wide range
examination was carried out to find DHSs specific
for Ig-b producing cells. The transcriptional enhancing
activities of the DHSs were examined by transient
transfection [30]. Three regions having transcriptional

enhancing activity were found in the intergenic region
between the Ig-b and GH genes. A member of the
OCT family transcription factors appeared to be
involved in the transactivation of the region which had
the highest enhancing activity of the three DHSs. The
state of acetylation in H3 and H4 histones and dime-
thylation in the H3 histone Lys4 residue were exam-
ined by chromatin immunoprecipitation (ChIP) [31].
The active promoter and cell type-specific DHS with
enhancing activity showed active histone modifications.
The sensitivity of chromatin to DNase I was measured
by real-time PCR (R-PCR) [16]. The regions with act-
ive histone modifications were again sensitive to
DNase I. To determine the in vivo role of DHSs in B
cell-specific transcription of the Ig-b gene, genetic stud-
ies such as transgenics and gene targeting are required.
Chicken B lymphocyte-derived DT40 cells are partic-
ularly useful for gene targeting because of the high rate
of homologous recombination [32,33]. Chicken Ig-b
gene is expressed in DT40 cells [34]. Previously, the
location of DHSs was surveyed in a 40 kb region
between 19 kb upstream and 21 kb downstream from
the transcriptional start site of the Ig-b gene [15].
Twelve DT40-specific DHSs were found: three in the
upstream Na channel gene, two between the Na channel
and Ig-b genes, one at the transcriptional start site,
one in the first intron of the Ig-b gene, four between
Ig-b and its downstream growth hormone (GH) genes,
and one in the downstream region of the GH gene.
Transcriptional enhancing activity, as determined by

transient transfection, was associated with DHSs in the
Na channel gene and in the first intron of the Ig-b
gene. Furthermore, the acetylation status of H3 and
H4 histones was examined [15]. In the present study,
10 DT40-specific DHSs found in the Ig-b locus were
divided into four groups based on location. For each
group, a deletion construct was introduced into DT40
cells. Ig-b gene expression, acetylation of H3 and H4
histones, and the remaining DHSs in the deleted cells
were examined to determine the roles of these DHSs
on the transcription of Ig-b gene and on the mainten-
ance of the active chromatin state.
Results
Generation of DT40/Cre cells with a long deletion
in one allele of the Ig-b locus
To genetically examine the mechanism of B cell-specific
transcription of the Ig-b gene, we generated DT40 ⁄ Cre
cells with a 16 kb deletion (16 kb Del) in one allele of
the Ig-b locus (Fig. 1). Introduction of the 16 kb Del
construct into DT40 ⁄ Cre cells resulted in two deletion
clones. In one of these two clones, the Bsr gene between
the loxP sequences was excised by Cre recombinase.
Following digestion of the genomic DNA with HindIII,
homologous recombination and the excision of Bsr gene
were confirmed by Southern hybridization using 0.9 kb
DNA ()10.1 kb to )9.2 kb) as the probe (Fig. 2). The
sizes of the resulting bands for each clone (Fig. 2B) were
the same as expected from the restriction map (Fig. 2A),
and thus homologous recombination followed by
excision of the Bsr gene was achieved.

Establishment of DT40/Cre cells expressing
small Ig-b mRNA
Initially, Ig-b protein appeared essential for the prolif-
eration of DT40 cells. To compensate for the absence
of Ig-b mRNA, a chicken b-actin promoter-driven Ig-b
gene whose transcript (1.5 kb) is shorter than wild type
(1.7 kb) due to deletion of the 3¢ untranslated region,
was introduced into 16 kb Del cells. From several
puromycin resistant clones, one clone containing wild
type and mutant Ig-b mRNA at a 2 : 1 ratio was selec-
ted and used for subsequent studies (Fig. 2C).
Generation of DT40/Cre clones deleted
in DT40-specific DHSs
Ten DT40-specific DHSs are present in the 16 kb region
of the Ig-b locus (Fig. 1) [15]. These DHSs were divided
into the following four groups (Fig. 1): region I, having
transcriptional enhancing activity [15] in the Na channel
gene ()7.8 kb to )6.0 kb; DHSs, )7.3 kb and )6.5 kb);
region II, promoter and upstream region of Ig-b gene
()2.7 kb to +0.05 kb; DHSs, )2.1 kb, )1.0 kb, and
0 kb); region III, in the first intron of the Ig-b gene
(+0.6 kb to +1.1 kb; DHS, +0.9 kb); region IV,
between Ig-b and GH genes (+4.5 kb to +6.7 kb;
DHSs, +4.8 kb, +5.2 kb, +5.8 kb, and +6.1 kb). Dele-
H. Matsudo et al. Deletion of hypersensitive sites in chicken Ig-b locus
FEBS Journal 272 (2005) 422–432 ª 2004 FEBS 423
tion constructs for regions I, II, and III were introduced
into the 16 kb Del cells with the extra Ig-b gene, and
between two and eight of the resulting clones were
found to have deletions for each of these regions as

judged by PCR. To remove the Bsr gene from these
clones by Cre recombinase, tentative recombinants
were treated with tamoxifen and then blasticidin sensi-
tive clones were obtained. In the course of the study,
Ig-b protein was found nonessential for the prolifer-
ation of DT40 cells (data not shown), and therefore
deletion of region IV was carried out by the introduct-
ion of the region IV deletion construct into 16 kb Del
cells.
Genomic Southern hybridization was performed to
confirm deletions (Fig. 3). For region I (Fig. 3A),
genomic DNA was digested with HindIII, and hybrid-
ized with a 0.9 kb probe for the Na channel gene; for
regions II (Fig. 3B) and III (Fig. 3C), DNA was cut
with Eco RI, and hybridized with Ig-b cDNA; for
region IV (Fig. 3D), SacI-digested DNA was hybrid-
ized with a probe for the GH gene. In each clone, the
sizes of the resulting bands that hybridized with the
probe coincided with that estimated from the restric-
tion map, and thus establishment of clones lacking
region I to IV was confirmed. A 1.3 kb band in
Fig. 3A, and a 3.1 kb band in Fig. 3D are derived
from the 16 kb Del allele, while the 6.6 kb band in
Fig. 3B,C is from the extra Ig-b gene.
Expression of Ig-b mRNA in DT40/Cre clones
deleted in DT40-specific DHSs
The level of Ig-b mRNA in cells with a deletion in
region I was the same as that in 16 kb Del cells
(Fig. 4A), and thus this region was shown to be nones-
sential for the transcription of Ig-b gene in DT40 cells.

Because region I enhances transcriptional activity when
linked to the Ig-b promoter and introduced into DT
40 cells [15], activity of this region in vivo was demon-
strated not to correlate with that observed in transient
transfection assays. Absence of Ig-b mRNA in cells
with a deletion in region II (Fig. 4A) demonstrated the
essential role of this region in the transcription of the
Ig-b gene. Deletion of region III decreased the expres-
sion of Ig-b mRNA to 40% of the expression in prede-
letion cells (Fig. 4A). Region III was found to enhance
Fig. 1. Organization of the chicken Ig-b locus and deleted regions. Exons are indicated by rectangles, and intron and intergenic regions by
solid lines. Horizontal arrows represent transcriptional orientations. Exon numbers are indicated. The hypothesized organization of a portion
of the Na channel gene for which the nucleotide sequence is unknown is represented by a dotted rectangle. Sizes of the intergenic regions
are shown in kb. Upward arrows indicate DNase I hypersensitive sites (DHSs), distances from the transcriptional start site of the Ig-b gene
in kb are indicated underneath. Black arrow, DT40-specific; gray arrow, LMH-specific; white arrow, common in both DT40 and LMH. DHS
arrows with enhancing activity are enclosed by rectangles. Positions of the eight targets for real-time PCR (R-PCR) are shown in the middle
[15]: 1, DHS )7.3 kb; 2, DHS )6.5 kb; 3, Na channel exon 24; 4, Ig-b promoter; 5, DHS +0.9 kb; 6, DHS +5.2 kb; 7, DHS +6.1 kb; 8, GH
exon 4. At the bottom, positions of the arm sequences used for the targeting constructs are shown by black bars and regions deleted by
dotted lines. Numbers above the 5¢-and3¢- ends of the region deleted are the distances from the transcriptional start site of the Ig-b gene.
Deletion of hypersensitive sites in chicken Ig-b locus H. Matsudo et al.
424 FEBS Journal 272 (2005) 422–432 ª 2004 FEBS
transcriptional activity in transient transfection four-
fold in DT40 cells [15], and thus, in contrast to region
I, in vivo activity of this region correlated with the
enhancing activity in transient transfection assays.
Region IV is located between the Ig-b and GH genes
and contains four DHSs within a 1.3 kb region. The
level of Ig-b mRNA in deletion cells was reduced to
56% of that in 16 kb Del cells (Fig. 4B). The proximal
part of this region (+4.6 kb to +5.5 kb containing

+4.8 kb and +5.2 kb DHSs) has no transcriptional
enhancing activity in transient transfection assays,
while the distal part (+5.6 kb to +6.4 kb including
+5.8 kb and +6.1 kb DHSs) decreases the transcrip-
tional activity of the Ig-b promoter to 50% of the
activity observed with the promoter alone. Thus, like
region I, in vivo activity of region IV did not correlate
with the activity observed in transient transfection
assays. These observations demonstrate that regions II,
III, and IV are involved in the transcription of the
Ig-b gene in DT40 cells, while region I is not. Further-
more, the enhancing activity of DHSs as determined
by transient transfection assays does not always corre-
late with the effect of DHS deletion on the expression
level of Ig-b mRNA.
Presence of DHSs in the Ig-b locus in cells
deleted in regions I–IV
DHSs in the Ig-b locus were examined by genomic
Southern hybridization in cells with region II deletion
(Fig. 5). DHSs located at )13 kb, )7.3 kb, and
)6.5 kb in the Na channel gene were present in the
region II deletion cells, similar to the parent cells
(Fig. 5A). DHSs located between the Ig-b and GH
genes, and DHSs at +10.3 kb and +13 kb positioned
downstream of the GH gene were both present in
region II deletion cells as well as in the predeletion
cells (Fig. 5C,D). Because the sequence from )2.7 kb
to +0.05 kb was lost in region II deletion cells, no
band corresponding to DHSs at )2.1 kb, )1.0 kb, and
0 kb was observed, while a DHS at +0.9 kb was pre-

sent (Fig. 5B). In summary, deletion of DHSs in
region II did not affect the presence of DHSs in the
Ig-b locus.
DHSs in region I, III, and IV deletion cells were also
examined. DHSs detected between )13 kb and +13 kb
in the Ig-b locus [15] were again present in all three
types of deletions although DHSs located at the dele-
tion region disappeared (data not shown), and thus
regions I, III, and IV were shown not to participate in
the maintenance of DT40-specific DHSs in the Ig-b
locus.
Acetylation of H3 and H4 histones in cells deleted
in region II
Acetylation of H3 and H4 histones is enhanced in and
around potentially active and transcribed genes
A
BC
Fig. 2. Construction of DT40 ⁄ Cre cells lacking the 16 kb region in
one Ig-b allele and expressing extra Ig-b mRNA shorter than the
wild type. (A) Organization of the Ig-b locus in DT40 ⁄ Cre cells lack-
ing the 16 kb region between )9.0 kb and +6.7 kb. 1, wild type
allele; 2, 16 kb deletion (Del) allele with blasticidin resistance (Bsr)
gene; 3, 16 kb Del allele without Bsr gene. Position of the probe
(0.9 kb; )10.1 kb HindIII to )9.2 kb AccI fragment) for hybridization
is indicated by a black rectangle. Size and position of the HindIII
fragment hybridizable with the probe is shown below the map.
Exons and introns are indicated as in Fig. 1. (B) Genomic Southern
hybridization. HindIII digested DNA (2.5 lg) was separated by
0.75% agarose gel-electrophoresis and hybridized with the probe
shown in (A). 1, DNA from wild type DT40 ⁄ Cre cells; 2, 16 kb Del

allele with Bsr gene; 3, 16 kb Del allele without Bsr gene. Restric-
tion fragment size (given in kb) was determined by kHindIII. Posi-
tions of the 1.3 kb, 2.7 kb, and 6.2 kb bands are shown in the right
margin. (C) Detection of the extra Ig-b mRNA by Northern hybridiza-
tion. Total RNA (3.0 lg) was prepared from cells containing the
expression vector for extra Ig-b mRNA and Northern hybridized
with Ig-b cDNA probe (nucleotides 312–1542; PmaCI ⁄ EcoRI) [34].
1, 16 kb Del allele without Bsr gene; 2, 16 kb Del allele without Bsr
gene and with extra Ig-b construct. Positions of 1.7 kb endogenous
and 1.5 kb extra Ig-b mRNA are shown in the right margin.
H. Matsudo et al. Deletion of hypersensitive sites in chicken Ig-b locus
FEBS Journal 272 (2005) 422–432 ª 2004 FEBS 425
A
B
C
D
Fig. 3. Confirmation of DHS deletions by Southern hybridization. Homologous recombination and the excision of Bsr gene by tamoxifen
treatment was first examined by PCR and then confirmed by Southern hybridization. Deleted regions: (A), I ()7.8 kb to )6.0 kb); (B),
II ()2.7 kb to +0.05 kb); (C), III (+0.6 kb to +1.1 kb); (D), IV (+4.5 kb to +6.7 kb). Left, gene organization; right, autoradiogram of the South-
ern hybridization. Position of the probes, the size and position of restriction fragments hybridizable with the probe, and exon ⁄ introns are
shown as in Fig. 2A. Positions of the DT40 specific-DHSs and the exon number are indicated as in Fig. 1. 1, wild type DT40 ⁄ Cre allele; 2,
each DHS deletion allele with Bsr gene; 3, each DHS deletion allele without Bsr gene. a, DNA from wild type DT40 ⁄ Cre cells; b, DNA from
cells with 16 kb Del allele (with extra Ig-b gene); c, DNA from cells with 16 kb Del allele (with extra Ig-b gene) and each DHS deletion allele
with Bsr gene; d, DNA from cells with 16 kb Del allele (with extra Ig-b gene) and each DHS deletion allele without Bsr gene. Probes: (A),
fragment from Na channel gene ()10.1 kb to )9.2 kb; HindIII ⁄ AccI); (B) and (C), Ig-b cDNA (nucleotides 312–1542; PmaCI ⁄ EcoRI) [34]; (D),
GH gene (+6.6 kb to +7.4 kb; NcoI ⁄ NcoI) [46]. Restriction enzyme digestion: A, HindIII; B and C, EcoRI; D, SacI. Restriction fragment size
(given in kb) was determined by kHindIII. Size of the bands observed in each autoradiogram is indicated at the right side. The 6.6 kb band
detected in lanes b, c, and d in autoradiogram (B), and lanes c and d in (C) is derived from the extra Ig-b gene. The 3.1 kb band found in
lanes b, c, and d in autoradiogram (D) is derived from 16 kb Del allele.
Deletion of hypersensitive sites in chicken Ig-b locus H. Matsudo et al.

426 FEBS Journal 272 (2005) 422–432 ª 2004 FEBS
[1,31,35–37]. Enhanced acetylation is mainly observed
at DHSs and their surroundings, although low level of
acetylation has been reported at DHSs such as HS-85
and 3¢ HS1 in the mouse b-globin locus [1]. Using
ChIP followed by R-PCR, the acetylation status of H3
and H4 histones in the chicken Ig-b locus has been
reported [15]. In DT40 cells, acetylation of H3 and H4
histones is enhanced at the DHSs ()7.3 kb, )6.5 kb,
0 kb, +0.9 kb, +5.2 kb, and +6.1 kb) deleted in this
study [15].
The acetylation status in region II deletion cells was
compared with that of predeletion cells (Fig. 6). A
prominent acetylation of both histones detected close to
the transcriptional initiation site (target 4) of the Ig-b
gene in wild type DT40 cells [15] was observed at the
same position both in predeletion cells and region II
deletion cells. Similar to wild type DT40 cells [15],
enhanced acetylation was demonstrated at the targets
closely positioned to DT40-specific DHSs in the Na
channel gene (close to DHSs at )7.3 kb and )6.5 kb;
targets 1 and 2), in the Ig-b first intron (DHS at
+0.9 kb; target 5), and between the Ig-b and GH genes
(DHSs at +5.2 kb and +6.1 kb; targets 6 and 7). At
these targets (targets 1, 2, 5, 6, and 7) in the region II
deletion cells, acetylation levels of both histones were
the same as that of the predeletion cells. In chicken
liver-derived LMH cells where no Ig-b mRNA is detec-
ted [15], and thus used as the negative control, no
enhanced acetylation of either histone was demonstrated

at the targets located close to the Ig-b promoter and
DT40-specific DHSs. Acetylation levels of both histones
at the targets in the last exon of the Na channel gene
(target 3) and in the fourth exon of the GH gene (target
8) was shown to be the same as in the cells before and
after region II deletion. In region II deletion cells, no
Ig-b mRNA was detected but the acetylation status of
both histones before and after deletion was demonstra-
ted to be the same, indicating that region II is essential
for B cell-specific transcription of the Ig-b gene but
unnecessary for the maintenance of the active chromatin
state in the Ig-b locus.
In transient transfection assays, the DNA fragment
containing a DHS at )2.1 kb shows no enhancing
activity of the Ig-b promoter in DT40 cells, while the
DHS at )1.0 kb decreases promoter activity to 50%
[15]. It would be interesting to learn whether the dele-
tion of DHSs at )2.1 kb and ) 1.0 kb has any effect
on the level of Ig-b mRNA; however, deletion of these
DHSs has been unsuccessful so far (data not shown).
Discussion
Ten Ig-b-producing cell-specific DHSs were grouped
into four regions, and each of these regions was dele-
ted in DT40 cells. The roles of the DHSs on Ig-b gene
expression and the maintenance of active chromatin
state in the Ig-b locus were examined in the dele-
tion cells. Establishment and maintenance of active
Fig. 4. Level of Ig-b mRNA in DT40 ⁄ Cre cells lacking DT40-specific DHSs. Using total RNA (3.0 lg) prepared from the deletion clones, the
level of Ig-b mRNA was examined by Northern hybridization with the Ig- b cDNA probe (nucleotides 312–1542; PmaCI ⁄ EcoRI) [34]. Intensity
of the bands was compared using

TYPHOON 9210 and IMAGE QUANT. (A) Positions of 1.7 kb endogenous and 1.5 kb extra Ig-b mRNA are
shown at the left and right, respectively. Lanes: RNA prepared from: 1, wild type DT40 ⁄ Cre cells (Wild type); 2, 16 kb Del allele (with extra
Ig-b gene) (Control 1); 3, 16 kb Del allele (with extra Ig-b gene) and the region I deletion allele (I Del); 4 and 5, 16 kb Del allele (with extra
Ig-b gene) and the region II deletion allele (II Del); 6, 16 kb Del allele (with extra Ig-b gene) and the region III deletion allele (III Del).
(B) Lanes: 1 and 2, 16 kb Del allele (without extra Ig-b gene) (Control 2); 3 and 4,16 kb Del allele (without extra Ig-b gene) and the region IV
deletion allele (IV Del). Probes: 1 and 3, human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA; 2 and 4, Ig-b cDNA.
H. Matsudo et al. Deletion of hypersensitive sites in chicken Ig-b locus
FEBS Journal 272 (2005) 422–432 ª 2004 FEBS 427
AB
C
D
Fig. 5. DNase I hypersensitive sites in the DT40 ⁄ Cre cells lacking region II. Isolated nuclei were treated with DNase I for 3 min at 20 °C.
Concentration of DNase I for treatment of nuclei from left to right: 75, 50, 25, 0 UÆmL
)1
. The DNA was purified from nuclei and digested
with EcoRI (A), ScaI(B),EcoRV (C) or EcoT22I (D). The digests were Southern hybridized with 0.9 kb ScaI ⁄ KpnI DNA ()4.6 kb to )3.7 kb;
nucleotides 849–1751 in accession number AB066568) (A,B) [34], 1.2 kb Ig- b cDNA (nucleotides 312–1542; PmaCI ⁄ EcoRI) [34] (C) or 0.2 kb
GH exon 5 (nucleotides 3515–3744 in D10484) (D) [46]. Restriction fragment size (given in kb) was determined by kHindIII. Analyzed region,
DHS, and position of the probe are shown at the bottom. The 2.5 kb band observed in (C) is derived from the extra Ig-b gene. Autoradio-
grams: left, wild type DT40 ⁄ Cre cells having 16 kb Del allele (with extra Ig-b gene); right, wild type DT40 ⁄ Cre cells having 16 kb Del allele
(with extra Ig-b gene) plus the region II deletion allele (mutant).
Deletion of hypersensitive sites in chicken Ig-b locus H. Matsudo et al.
428 FEBS Journal 272 (2005) 422–432 ª 2004 FEBS
chromatin is essential for transcription of cell type-spe-
cific genes. Active chromatin is characterized by an
open chromatin structure that is generally sensitive to
DNaseI, presence of cell type-specific DHSs [7,8], and
active-type modification of core histone tails [9–11].
From the earliest stage of the study [6], DNase I gen-
eral sensitivity has been demonstrated to be a good

parameter to determine chromatin state [1–5]. Distinct
differences are often obtained between positive and
negative controls in DHS and histone modification
analyses; in contrast, these differences are often much
smaller in DNase I general sensitivity analysis.
Although DNase I general sensitivity analysis of the
chicken Ig-b locus has not been finished because of the
technical difficulty of this type of assay, there may be
no difference in the range and extent of general sensi-
tivity to DNase I with or without DHSs deleted in this
study because the positions of DHSs remain the same.
In the chicken b-globin locus, the DNase I sensitive
region coincides with the region in which the core
histones are hyperacetylated [3,38]. In the mouse b-glo-
bin locus, hyperacetylated H3 and H4 histones are
located in a much wider DNase I sensitive region
[1,12]. Thus, the DNase I sensitive chromatin state is
not always associated with active-type modification of
core histones. Furthermore, a cell type-specific DHS,
HS-85, in the mouse b-globin ⁄ odorant receptor locus is
located in a region with a low acetylation level of the
core histones and a chromatin state in the flanking
region resistant to DNase I [1]. In this case, the forma-
tion of cell type-specific DHS again does not occur in
the context of a DNase I sensitive chromatin region,
or a region with active-type histone modifications.
Transgenic mice with a 99 bp deletion (containing
two Pit-1 binding sites) of the DHS I region which is
located within the locus control region (LCR) in the
human GH locus, show loss of H3 and H4 acetyla-

tion ranging from 32 kb upstream of the transcrip-
tional start site of the GH gene to the GH gene [18].
Thus, the deleted sequence is essential for the estab-
lishment and maintenance of acetylation in the GH
locus. Because deletion of this region has no effect on
the formation of cell type-specific DHSs, the region
required for H3 and H4 acetylation does not neces-
sarily coincide with that involved in the DHS forma-
tion. Deletion of DHS1–6, the entire mouse b-globin
LCR, results in extraordinarily low levels of b-globin
mRNA. However, both the acetylation state of the
promoter region of the active b-globin gene [19] and
the DNase I general sensitivity in the b-globin locus
are the same as the wild type control [17]. Thus,
there again has been no description either of the
region involved in DNase I general sensitivity or
of the region for the formation of DHSs, although
chromatin opening activity of the LCR has been des-
cribed in several loci using transgenic mouse models
[29,39–42].
Because the chromatin state of the cells with any
deletion in the regions I–IV in the Ig-b locus, judged
by DHSs as the indicator, was the same as that in pre-
deletion cells, the region required for the maintenance
of the active chromatin state is presumed to be present
at the DHSs located further upstream or downstream
of the DHSs examined in this study. Two DT40-speci-
fic DHSs, one at )13 kb in the Na channel gene and
the other at +13 kb downstream of the GH gene, have
Fig. 6. H3 and H4 acetylation of the Ig- b locus in the region II dele-

tion cells. The acetylation of wild type DT40 ⁄ Cre cells with 16 kb
Del allele (with extra Ig-b gene), wild type DT40 ⁄ Cre cells with
16 kb Del allele (with extra Ig-b gene) plus the region II deletion
allele, and LMH cells is indicated by the black, gray and white bars,
respectively. (Top) H3 acetylation. (Middle) H4 acetylation. Acetyla-
tion values are shown by fold enrichment compared with untreated
control DNA as reported previously [15,31]. Three amplifications
were performed for each target. The values of the ng control DNA
equivalents were estimated using standard calibration, and fold
enrichment was determined from these values [31]. Values are
mean ± SE. An over-scaled bar is shown by double wavy lines, and
its mean value is shown at the left. Bottom; the positions of eight
targets are shown. Exon ⁄ introns are shown as in Fig. 1. Arrows,
DHSs; +, transcriptional start site.
H. Matsudo et al. Deletion of hypersensitive sites in chicken Ig-b locus
FEBS Journal 272 (2005) 422–432 ª 2004 FEBS 429
already been described [15], and deletion of these
DHSs should be performed in the future.
Transcription of the adult-type mouse b-globin gene
is suggested to require the assembly of DHSs into one
complex named the active chromatin hub at around
the transcriptional start site. This complex includes six
DHSs in the LCR located 40–60 kb upstream from the
active gene, DHSs present in the odorant receptor
genes found 40 kb further upstream from the LCR,
and those present in 20 kb downstream from the b-glo-
bin gene [43]. Combined deletion of the separate DHS
groups would be necessary in future studies because
the deletion of a single region showed no remarkable
effect on the chromatin state of the Ig-b locus.

The activities of DHSs in the Ig-b locus examined
by transient transfection [15] did not correlate with
those determined by deletion in regions I and IV. Sim-
ilar situations have been reported at DHSs within the
human b-globin LCR [44]. Although the reason for the
inconsistency of the DHS activity between transient
transfection and in vivo deletion is unknown, the phe-
notype observed in deletion cells is likely to reflect the
in vivo role of the DHS because genomic DNA is dele-
ted from its native context. Another reason may be the
redundancy between different elements in the Ig-b
locus.
Materials and methods
Cells
Liver-derived LMH cells were obtained from the Japanese
Cancer Research Resources Bank (Tokyo, Japan).
DT40 ⁄ Cre cells [45] that produce MerCreMer protein, a Cre
recombinase with hormone binding domains of the mutant
mouse estrogen receptor (Mer) on both ends, which localizes
in the cytoplasm in the absence of estrogen derivative,
4-hydroxy tamoxifen. Cells were propagated in RPMI1640
(Nissui, Tokyo, Japan) ⁄ 10% fetal bovine serum (JRH Bio-
sciences, Lenexa, KS, USA) ⁄ 1% chicken serum (GibcoBRL,
Grand Island, NY, USA) at 39.5 °C for DT40 ⁄ Cre and
37 °C for LMH. DT40 ⁄ Cre cells were grown with
2mgÆmL
)1
of geneticin (GibcoBRL) which is required for
establishment of DT40 ⁄ Cre cells. For selection, blasticidin
(Funakoshi, Tokyo, Japan) and puromycin (Sigma-Aldrich,

St. Louis, MO, USA) were used at 30 lgÆmL
)1
and
0.5 lgÆmL
)1
, respectively.
Preparation of targeting constructs and extra Ig-b gene
expression construct pLoxBsr [45] containing the chicken
b-actin promoter-driven blasticidin resistance (Bsr) gene
with mutant loxP sequences at both ends was used as a tar-
geting vector. For the targeting construct, genomic DNA of
about 2 kb in size was linked on both sides with the actin
promoter-driven Bsr gene. Positions of left and right arms
of the five constructs (16 kb Del, I to IV; Fig. 1) are as fol-
lows (the transcriptional start site of Ig-b gene is shown as
base number one): 16 kb Del-L ()11.4 kb to )9.0 kb,
SacI ⁄ MluI; 2.4 kb), 16 kb Del-R (+6703 to +8768;
2066 bp), I-L ()10.1 kb to )7.8 kb, BamHI ⁄ XhoI; 2.3 kb),
I-R ()6009 to )3942; 2068 bp), II-L ()4613 to )2692;
1922 bp), II-R (+50 to +1959; 1910 bp), III-L ()1369 to
+578; 1948 bp), III-R (+1079–3130; 2052 bp), IV-L
(+2721 to +4524, 1804 bp), IV-R (same as 16 kb Del-R).
Based on the reported nucleotide sequences [15,34,46], arm
DNAs were amplified with Pyrobest polymerase (Takara
Bio, Kyoto, Japan), and ligated into the vector. Restriction
fragments were used for the arms located in the unsequ-
enced region. The Bsr gene was inserted in the same orien-
tation as the flanking gene. DT40 ⁄ Cre cells were
electroporated with linearized DNA and clones were
obtained as described [47].

Using cloned chicken Ig-b cDNA as the template, 0.9 kb
DNA (nucleotide number 20–930) [34] was amplified by
Pyrobest polymerase and cloned into HindIII ⁄ NheI sites of
the pExpress vector [45]. The chicken b-actin promoter-
driven puromycin resistance (Puro) gene was inserted into
the XhoI site of the construct.
Selection of homologous recombinant clones
by PCR and tamoxifen treatment
To determine homologous recombination, PCR was carried
out as described [45] using the following primers: 5¢-
CGATTGAAGAACTCATTCCACTCAAATATACCC-3¢
(in Bsr gene) [45]. Primers (30-mer) in the genome were set
up at 10–100 bases downstream from the right arm: 5¢-
TAGTTTCTCAAACACTCTGTCTGAGGTGCC-3¢ (16 k
Del; +8778 to +8807); 5¢-ATGGGTTCATAGGAGACC
TTTGAGGGGTTG-3¢ (I: )3782 to )3811); 5¢-TAGATG
CCGTTGTCCTCGTAGCTGATCCTG-3¢ (II: +2085 to
+2114); 5¢-AGTGATGTCCTCGTAGGTGGCAATCTGC
TC-3¢ (III: +3438 to +3467) (IV: same as 16 k Del).
Clones whose DNA contained about 3 kb predictable
sequences by PCR were selected as tentative clones with
homologous recombination.
DT40 ⁄ Cre clones with homologous recombination were
treated with 0.1 mm 4-OH-tamoxifen (Sigma, St. Louis,
MO, USA) for 24 h and then cloned in 96 well microtiter
plates. Deletion of the Bsr gene was confirmed by genomic
Southern hybridization.
DNase I digestion, hybridization, and histone
acetylation analysis
For DNase I hypersensitivity analysis, nuclei were prepared

and treated with DNase I (Takara Bio) and DNA was pre-
pared as described [30]. Restriction enzyme digestion and
Deletion of hypersensitive sites in chicken Ig-b locus H. Matsudo et al.
430 FEBS Journal 272 (2005) 422–432 ª 2004 FEBS
Southern hybridization were carried out as reported previ-
ously [48]. Probe DNA was labeled by the random-priming
methods using [
32
P]dCTP[aP]. The following probes were
used: Ig-b cDNA (nucleotides 312–1542, 1231 bp) [34], Na
channel gene ()10.1 kb to )9.2 kb, HindIII ⁄ AccI; 0.9 kb),
GH gene (+6560 to +7380, NcoI ⁄ NcoI; 821 bp) [34]. Pre-
paration of total RNA followed by Northern hybridization
was performed as described previously [31]. A human glyc-
eraldehyde-3-phosphate dehydrogenase cDNA probe was
obtained from Clontech (Palo Alto, CA, USA). The inten-
sity of bands on the Northern hybridization was compared
by typhoon 9210 and image quant (Amersham Bioscienc-
es, Piscataway, NJ, USA). Acetylation status of H3 and H4
histones was examined by chromatin immunoprecipitation
(ChIP) and real-time PCR (R-PCR) as described previously
[15].
Acknowledgements
We thank the Japanese Cancer Research Resources
Bank for providing cells. This work was supported by
Rikkyo University for the Promotion of Research.
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Deletion of hypersensitive sites in chicken Ig-b locus H. Matsudo et al.
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