MOLECULAR AND CELLULAR BIOLOGY, May 2010, p. 2473–2484
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 30, No. 10
Expression of a Testis-Specific Form of Gal3st1 (CST), a Gene
Essential for Spermatogenesis, Is Regulated by the CTCF
Paralogous Gene BORIS?†
Teruhiko Suzuki, Natsuki Kosaka-Suzuki, Svetlana Pack,* Dong-Mi Shin, Jeongheon Yoon,
Ziedulla Abdullaev, Elena Pugacheva, Herbert C. Morse III,
Dmitri Loukinov, and Victor Lobanenkov*
Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, NIH, Rockville, Maryland
Received 17 August 2009/Returned for modification 2 October 2009/Accepted 2 March 2010
Previously, it was shown that the CTCF paralogous gene, BORIS (brother of the regulator of imprinted sites)
is expressed in male germ cells, but its function in spermatogenesis has not been defined. To develop an
understanding of the functional activities of BORIS, we generated BORIS knockout (KO) mice. Mice homozy-
gous for the null allele had a defect in spermatogenesis that resulted in small testes associated with increased
cell death. The defect was evident as early as postnatal day 21 and was manifested by delayed production of
haploid cells. By gene expression profiling, we found that transcript levels for Gal3st1 (also known as
cerebroside sulfotransferase [CST]), known to play a crucial role in meiosis, were dramatically reduced in
BORIS KO testes. We found that CST is expressed in testis as a novel testis-specific isoform, CST form FTS,
that has a short exon 1f. We showed that BORIS bound to and activated the promoter of CST form FTS.
Mutation of the BORIS binding site in the promoter reduced the ability of BORIS to activate the promoter.
These findings define transcriptional regulation of CST expression as a critical role for BORIS in
CTCF is a ubiquitously expressed chromatin factor contain-
ing an 11-zinc-finger (11-ZF) DNA binding domain that is
highly conserved from Drosophila melanogaster to humans (39,
43, 61). Identities among vertebrate orthologs of 90 to 99%
imply critical functions for the protein now known to include
regulation of gene expression and chromatin organization (10,
28, 41). CTCF was originally identified as a repressor of Myc
but was later shown to function in regulation of transcription
when bound to promoters of specific genes such as APP, Rb,
p53, hTERT, and ARF (9, 11, 12, 29, 35, 46, 49, 54, 57). In
addition, CTCF is also known to act as a chromatin insulator,
which blocks inappropriate activation or inactivation of genes
by flanking regulatory elements (39). A constitutive insulator
function of CTCF was originally identified in a reporter con-
struct based on the HS4 element cloned from the chicken
?-globin locus (3, 21), while later studies revealed that CTCF
also mediate methylated CpG-sensitive insulation of imprinted
genes (24). CTCF was found to bind unmethylated sequences
in the maternal allele of the H19 imprinting control region
(H19 ICR) as a part of a complex three-dimensional loop
structure that partitions the enhancer from IGF2, resulting in
inhibition of expression. CTCF could not bind methylated H19
ICR sequences of paternal origin, resulting in loss of enhancer
blocking activity and activation of IGF2 expression (14, 33).
More recently, CTCF sites have been found to maintain epi-
genomic integrity of unstable repeats (34) and latency control
of herpes simplex virus (HSV), Epstein-Barr virus (EBV), and
Kaposi’s sarcoma-associated herpesvirus (2, 5, 50).
The gene BORIS (brother of the regulator of imprinted
sites), also known as CTCFL (CTCF-like) and CTCF-T (CTCF
testis specific), has been identified as a mammalian paralog of
CTCF (28, 37). Recent studies showed that a BORIS ortholog
is present in the platypus, reptiles, and higher organisms (20).
Evolutionarily, expression of BORIS became progressively re-
stricted to the testis, whereas CTCF is ubiquitously expressed
in all species tested. This implies that BORIS has acquired
testis-specific functions in mammalian organisms.
In addition to its normal expression in the testis, recent
studies revealed that various tumors and cancer cell lines are
also BORIS positive, with frequent coexpression of cancer
testis antigens (CTA) (7, 17, 48, 52, 55). In addition, it was
shown that conditional expression of BORIS induced expres-
sion of a series of CTA genes, including MAGE-A1, NY-
ESO-1 (17, 52), and SPANX (30). This suggests that BORIS
may normally act to regulate expression of CTA genes, al-
though some data suggest otherwise (25).
Recent genome-wide analyses of CTCF binding sites by
chromatin immunoprecipitation (ChIP)-chip or ChIP-se-
quencing analysis identified approximately 14,000 or more
CTCF binding sites throughout the genome (6, 27). Kim et al.
(27) documented that almost 80% of the binding sites shared
* Corresponding author. Mailing address for Victor Lobanenkov:
Molecular Pathology Section, Laboratory of Immunopathology, Na-
tional Institutes of Allergy and Infectious Diseases, NIH, Twinbrook I,
Room 1417, MSC-8152, 5640 Fishers Lane, Rockville, MD 20852.
Phone: (301) 435-1690. Fax: (301) 402-0077. E-mail: vlobanenkov
@niaid.nih.gov. Present address for Svetlana Pack: Chromosome
Pathology Unit, Laboratory of Pathology, CCR, National Cancer
Institute, NIH, 10 Center Drive, Rm. 2N115, MSC1500, Bethesda, MD
20892. Phone: (301) 451-2711. Fax: (301) 402-0043. E-mail: spack
† Supplemental material for this article may be found at http://mcb
?Published ahead of print on 15 March 2010.
consensus recognition sequences with evolutionary conserva-
tion. It is important to recognize that in contrast to the 11-ZF
domain, the N- and C-terminal regions of BORIS share no
homology with similarly placed CTCF sequences. This suggests
that BORIS may exhibit functions distinct from CTCF based
upon partner interactions that are dependent on these unique
domains. In fact, BAT3 and PRMT7 were found to interact
with BORIS through the unique N-terminal domain, implying
that BORIS is functionally distinct from CTCF despite sharing
DNA recognition sequences (23, 40).
Production of seminolipid, a primary glycolipid in the testis,
is catalyzed by cerebroside sulfotransferase (CST), which is
encoded by Gal3st1 through sulfation of galactosylalkylacyl-
glycerol (19, 53). The importance of CST and its product in
spermatogenesis was revealed through analyses of CST knock-
out (KO) mice, which displayed male-specific sterility due to a
spermatogenesis defect apparent before the first meiotic divi-
sion (18, 60). Mouse CST has eight splicing variants, all of
which have the same coding region with different 5?-untrans-
lated region (5?-UTR) sequences (16). Although different tis-
sues express various combinations of CST splicing variants,
testis expresses only CST form F, implying that there are testis-
specific transcription factors that activate the promoter of this
unique isoform. In view of the crucial function of CST in
spermatogenesis, regulation of the CST form F promoter
should be a critical determinant for proper spermatogenesis.
In this study, we analyzed BORIS KO mice to develop a firm
understanding of the physiological functions of BORIS. We
found that BORIS?/?mice exhibited a spermatogenesis defect
at meiosis. We identified a novel testis-specific CST splicing
variant, CST form FTS, for which expression is dramatically
suppressed by the loss of BORIS. Our findings suggest that
BORIS plays an important function in spermatogenesis
through regulation of CST expression.
MATERIALS AND METHODS
Antibodies. A glutathione S-transferase (GST)-tagged mouse BORIS C-ter-
minal fragment containing amino acid residues 538 to 636 was prepared from
Escherichia coli and used to immunize two rabbits. Serum antibodies were affinity
purified on a GST-BORIS C-terminal fragment column, and then GST-reactive
antibodies were depleted with a GST column. Antibodies were produced and
purified by GenScript (Piscataway, NJ).
Targeted disruption of the BORIS gene. BORIS knockout mice were generated
at Ozgene (Ozgene Pty. Ltd., Bentley, Western Australia, Australia). The tar-
geting vector was designed to replace the BORIS gene from the first methionine
to exon 8 with an eGFP reporter casette flanked by LoxP/Lox511 sequences and
a PGK-neomycin cassette flanked by FLP recombinase target (FRT) sequences.
Primers used for targeting vector construction, genotyping, and generation of
probes for Southern blotting are listed in Table S1 of the supplemental material.
The targeting vector was linearized and electroporated into 129S1/Sv-derived
W9.5 embryonic stem (ES) cells. Targeted ES cells were injected into blastocysts
from C57BL/6 (B6) mice. Chimeric male mice were crossed with C57BL/6
females, and the BORIS?/?mice obtained were mated to obtain BORIS null
mice. The PGK-neomycin cassette was deleted by crossing with B6.Cg-
Tg(ACTFLPe)9205Dym/J mice (The Jackson Laboratory, Bar Harbor, ME).
The use of mice in this study was approved by the NIAID Animal Care and Use
Committee under protocol LIP-5.
Histochemistry. Testes were fixed with 10% formalin and embedded in par-
affin. Five-micrometer sections were prepared for histological analysis. Terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)
staining was performed using the DeadEnd colorimetric TUNEL system (Pro-
mega, Madison, WI) according to the manufacturer’s protocol. TUNEL-positive
cells in a field, which corresponded to approximately 0.6 mm2, were counted to
quantify apoptotic cells in testes. Three mice were examined for each genotype.
DNA flow cytometry. DNA flow cytometry was performed as described previ-
ously with slight modifications (31). Briefly, testicular cells that were fixed with
70% ethanol at least overnight were treated with 0.5% pepsin solution for 75 min
at 37°C followed by treatment with phosphate-buffered saline (PBS) supple-
mented with 80 ?g/ml RNase and 1% fetal bovine serum (FBS) for 20 min at
room temperature. Cells were then stained with PBS containing 5 ?g/ml pro-
pidium iodide (PI) and 1% FBS for 30 min on ice. Stained cells were analyzed
with a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ).
Purification of spermatocytes and round spermatids. Spermatocytes and
round spermatids were purified by centrifugal elutriation (38) followed by flow
cytometric sorting of cells stained with Vybrant DyeCycle Green (Invitrogen,
Carlsbad, CA) to obtain cell fractions with high purity. Briefly, decapsulated
testes were treated with collagenase followed by treatment with trypsin, and the
dissociated cells were used for centrifugal elutriation. Partially purified sper-
matocytes and the round spermatids fractions were incubated with 10 ?M
Vybrant DyeCycle Green for 30 min at 32°C followed by 4,6-diamidino-2-phe-
nylindole (DAPI) staining. Cells were then sorted on a FACSAria (Becton
Dickinson) to purify spermatocytes and round spermatids. DAPI-positive dead
cells were eliminated. The purity of cells was confirmed by flow cytometry analysis
round spermatids, 92.2% ? 2.5% [means ? standard deviations]).
Surface spread staining. Surface spreads of spermatocytes were prepared as
described previously (42). Samples were stained with anti-SCP3 (Novus Biologi-
cals, Littleton, CO) and anti-?H2AX antibodies (Millipore, Billerica, MA) for
1 h at room temperature followed by staining with Alexa Fluor 488- or Cy3-
conjugated secondary antibody for 1 h at room temperature. Samples were
counterstained with DAPI.
Microarray analysis. Microarray chips printed by the NIAID Microarray Re-
search Facility comprised approximately 18,000 genes represented by 70-mer
oligonucleotides. We studied five sets of wild-type and BORIS?/?testes pre-
pared from postnatal day 14 (P14) mice. After the raw data were normalized with
the Lowess smoothing function, the significant genes were identified with signif-
icance analysis of microarrays (51) with a false discovery rate of 3%, followed by
a selection of genes with changes greater than twofold. Expression of genes with
significant changes by microarray analyses was validated by quantitative PCR
(qPCR). Microarray data were deposited to the NCBI Gene Expression Omni-
bus database (accession number GSE19162).
RT-PCR and qPCR. Total RNA was prepared using an RNeasy minikit (Qia-
gen, Valencia, CA). cDNA was prepared using the SuperScript III first-strand
synthesis system (Invitrogen) according to the manufacturer’s protocol. Quan-
tiative PCR (qPCR) was performed using the SYBR green PCR master mix
(Applied Biosystems, Foster City, CA) and the 7900HT sequence detection
system (Applied Biosystems). Primers used for conventional reverse transcrip-
tion-PCR (RT-PCR) and quantitative PCR are listed in Table S1 in the supple-
mental material. Primers for qPCR were designed using the Primer Express
software (Applied Biosystems). Expression levels were normalized against the
housekeeping gene Gapdh. For ?Ctcalculations, a Ctvalue of 40 was used for
samples that had Ctvalues over 40. Absolute quantification was performed to
analyze the transcription level of CST form FTSand FSSin CST form FTotalby
using a plasmid that contained the partial cDNA sequence of CST form FSS
cloned from stomach cDNA as a standard. The numbers of samples prepared
from individual mice for qPCR are described in the figure legends. Each sample
subjected to qPCR was analyzed in triplicate. Student’s t test was performed to
evaluate statistical significance. Data shown are means ? standard deviations.
5?-RACE. Total RNA prepared from 3-month-old mouse testes was subjected
to 5? rapid amplification of cDNA ends (5?-RACE) analysis utilizing the Gene-
Racer kit (Invitrogen), which ensures amplification of only full-length transcripts
by eliminating truncated messages from the amplification process, according to
the manufacturer’s instructions. Reverse transcription was performed using a
CST-specific reverse primer, 5?-CCATTGGGGAAAGCGAACTTGAG-3?, fol-
lowed by nested PCR using a reverse primer specific for CST, 5?-AGTGTGCT
GCTGGCGGTCTTGTG-3?. Both CST-specific primers were designed on the
open reading frame of CST, which is common for all isoforms of CST. Purified
PCR fragments were cloned in a pGEM-T vector (Promega), sequenced
(Genomics Research Facility, Rocky Mountain Laboratories, NIAID, NIH,
MT), and analyzed.
Bisulfite sequencing. Bisulfite modification of genomic DNA prepared from
kidney or spermatocytes was performed using an Imprint DNA modification kit
(Sigma-Aldrich, St. Louis, MO). A 314-bp sequence was amplified using 5?-GG
GAAAGTTTTTGTATTATTGTATGAT-3? and 5?-AACTAACTCTCTACTA
AACCTAAAAACC-3? as primers with PCR Platinum Taq polymerase under
the following conditions: 94°C for 2 min; 35 cycles of 94°C for 1 min, 52°C for
30 s, and 72°C for 1 min; and 72°C for 5 min. The amplicon was ligated into the
2474SUZUKI ET AL.MOL. CELL. BIOL.
pGEM-T vector. Following transformation, plasmids from individual bacterial
colonies were isolated and subjected to sequencing.
EMSA and methylation interference assay. An electrophoretic mobility shift
assay (EMSA) and methylation interference assay were performed as described
previously (13, 44). Briefly, proteins for the assays were synthesized using plas-
mid pET-16b (Novagen, Madison, WI) with inserts of BORIS, CTCF, CTCF
zinc-finger domain (11-ZF) sequences, or luciferase T7 control DNA with the
TnT in vitro transcription-translation system (Promega). One primer for each
DNA fragment was labeled with [?-32P]ATP and used for PCR to prepare DNA
fragments. Amplicons were gel purified and used for the assays. For supershift
assays, proteins and DNA fragments were preincubated for 30 min at room
temperature for complex formation followed by incubation with antibodies for 20
min. A mixture of two rabbit anti-mouse BORIS antibodies and a mixture of
mouse monoclonal antibodies against CTCF (44) were used for supershift assays.
Samples were loaded onto 5% native polyacrylamide gels in 0.5?Tris-borate-
EDTA. The supershifted band of BORIS was found at the top of the gel due to
the polyclonality of the anti-mouse BORIS antibodies.
Luciferase assay, cells, and transfection. Fragments containing the entire
5?-UTR sequences of CST form FTSand its promoter sequences starting from
promoter position ?179, ?359, or ?515 were cloned into pGL3-basic vector
(Promega). NIH 3T3 cells cultured in Dulbecco’s modified Eagle’s medium
supplemented with 10% FBS and penicillin-streptomycin were transfected using
Fugene 6 (Roche, Indianapolis, IN) according to the manufacturer’s protocol
with luciferase constructs together with internal control vector and BORIS or
CTCF expression vectors cloned into pCIneo where indicated. Cells were cul-
tured for 48 h at 37°C with 5% CO2. Luciferase assays were performed using a
Dual-Luciferase reporter assay system kit (Promega) according to the manufac-
turer’s protocol. Three independent samples were analyzed for each experiment,
and the numbers of experiments are described in the figure legends. Student’s t
test was performed to evaluate statistical significance. Luciferase activity data
shown are the means ? standard errors of the means.
ChIP assay. ChIP assays were performed based on the Upstate Biotechnology
protocol. Briefly, decapsulated testes were washed with PBS and minced with a
scalpel. Samples were fixed with 1% formaldehyde for 15 min with rocking
followed by two washes with PBS. Fixed samples were homogenized with a
Dounce homogenizer, and pelleted samples were suspended in SDS lysis buffer
(20 mM Tris-HCl [pH 8], 0.1% SDS, 2 mM EDTA, 150 mM NaCl, 1% Triton
X-100) supplemented with a protease inhibitor cocktail. Lysates corresponding
to 30 mg of tissue were used for a ChIP assay. A mixture of rabbit anti-mouse
BORIS antibodies was used for ChIP. Preimmune sera were used as a control.
Primers used for quantitative PCR are listed in Table S1 of the supplemental
Targeted disruption of the BORIS gene. To identify the
physiological function of BORIS and its target genes, we gen-
erated BORIS knockout mice. The BORIS gene, from the first
methionine to exon 8, which includes ZF 1 to 10 and the
N-terminal half of ZF11, was replaced with GFP and a neo-
mycin cassette (Fig. 1A). We confirmed the recombination by
Southern blotting (Fig. 1B), genomic PCR (Fig. 1C), RT-PCR
(Fig. 1E), and quantitative RT-PCR (Fig. 1F), as well as the
generation of mice with the PGK-neomycin cassette removed
[BORIS?/?(-neo)] (Fig. 1D). Expression of CTCF did not
change with the deletion of BORIS (Fig. 1G). By RT-PCR, we
detected expression of GFP only in testis (Fig. 2), consistent
with the normal expression pattern of BORIS transcripts; how-
FIG. 1. (A) Targeting scheme for generation of BORIS (Ctcfl) KO mice. Deletion of the BORIS gene proceeds from the first methionine to
exon 8, which includes ZF-1 to -10, and the N-terminal half of ZF-11 is replaced with GFP and a neomycin cassette. The probe used for Southern
blotting is indicated. Filled boxes indicate open reading frames; red boxes indicate the coding regions of the 11-ZF domain; open boxes indicate
untranslated regions; the open triangle indicates a LoxP site; the filled triangle indicates a Lox511 site. FRT sequences are shown with red triangles.
B, BglII. (B) Southern blotting. Genomic DNA was digested with BglII and hybridized with a probe flanking the targeting construct at the 3? end
as diagramed in panel A. (C) Genomic PCR typing of BORIS?/?mice. (D) Genomic PCR typing of BORIS?/?(-neo) mice. (E) cDNAs prepared
from testes of 3-month-old mice were subjected to RT-PCR. (F and G) cDNAs prepared from testes of 3-month-old mice were quantified by qPCR
for expression of transcripts for BORIS (F) and CTCF (G) (n ? 3). Expression levels are shown as the ratio to wild type.
VOL. 30, 2010 BORIS REGULATES SPERMATOGENESIS THROUGH CST2475
ever, we could not detect GFP expression by fluorescence
microscopy or flow cytometry (data not shown), probably due
to low expression of GFP.
Spermatogenesis defect in BORIS?/?mice. Matings of
BORIS?/?mice yielded progeny at the expected Mendelian
ratios (data not shown). As BORIS is exclusively expressed in
testis (37) (Fig. 2), we focused our studies on testicular devel-
opment and function. Although the BORIS?/?male mice were
fertile (Table 1), we found that BORIS?/?testes were signifi-
cantly smaller than those of their wild-type counterparts (Fig.
3A and B), indicating that BORIS had important functions in
testicular development. A reduction in testicular size for
BORIS?/?mice could be detected as early as P28 (Fig. 3B). To
determine the basis for this difference, we performed histologic
studies of testes from P28 BORIS?/?mice. Testes from
BORIS?/?mice had reduced cellularity compared to testes of
wild-type mice (Fig. 3A). Furthermore, testes from BORIS?/?
mice had multinucleated cells (Fig. 3A, arrows), a feature
found in a number of spermatogenesis-defective mice. We
confirmed that BORIS?/?(-neo) mice also displayed a similar
defect (Fig. 3A, right column, lower panel) and that they were
fertile as well (data not shown). These results indicate that the
spermatogenesis defect was due specifically to the loss of
BORIS and was not affected by the presence of the PGK-
neomycin cassette. To analyze whether the reduced cellular-
ity of BORIS?/?testis might be due to increased cell death,
we performed TUNEL staining and found that the fre-
quency of apoptotic cells was greatly increased in BORIS?/?
testis compared to wild-type testis (Fig. 3C). This result
suggests that the smaller testes of BORIS?/?mice could be
ascribed at least in part to increased cell death with effects
To further analyze the effects of BORIS deficiency on the
progression of spermatogenesis, we performed DNA flow cy-
tometry on testicular cells. This allowed us to quantify sub-
populations of testicular cells: tetraploid cells (primarily sper-
matocytes), diploid cells (a mixture of spermatogonia, secondary
spermatocytes, and somatic cells), and haploid cells (sperma-
tids). We found a deficiency in production of haploid cells in
BORIS?/?testis compared to wild-type testis as early as P21
(17.8% ? 5.2% [wild type] versus 9.5% ? 5.2% [BORIS?/?];
P ? 0.05) as well as at P28 (48.8% ? 2.3% [wild type] versus
28.9% ? 6.6% [BORIS?/?]; P ? 0.05) (Fig. 3D). The fact that
there was no significant difference in testicular weight at P21
was probably due to the scarcity of haploid cells at this stage of
development, the beginning step in haploid cell production. To
analyze the effect of BORIS on synapsis, we performed surface
spread staining of spermatocytes. Although XY bodies positive
for ?H2AX were formed in spermatocytes of both BORIS?/?
and wild-type mice, around 20% of BORIS?/?spermatocytes
showed abnormal aggregation of the synaptonemal complex
protein, SCP3, which does not coincide with ?H2AX (Fig. 3E).
This finding is similar to that of the abnormal structure re-
ported in Dnmt3L-deficient spermatocytes (4). These results
indicate that abnormalities in spermatogenesis in BORIS?/?
mice begin around P21 and are evidenced by a significant delay
and defect in meiosis.
Loss of BORIS is associated with reduced expression of
CST. We reasoned that alterations in gene expression respon-
sible for the spermatogenesis defect found in BORIS?/?mice
would be evident before day 21, when the defect could first be
detected. To examine this possibility, we performed gene ex-
pression profiling using oligonucleotide microarrays to study
testes prepared from P14 mice, an age at which we found no
significant changes in the population of spermatogenic cells
(wild type 2n, 70.6% ? 4.8%; wild type 4n, 20.7% ? 5.7%;
BORIS?/?2n, 74.7% ? 4.2%; BORIS?/?4n, 18.7% ? 3.6%)
(Fig. 3D). We identified 24 genes showing statistically signifi-
cant differences in expression, and 4 of these genes showed
more-than-2-fold differences in expression levels (see Fig. S2 in
the supplemental material). To validate the differential expres-
sion, we employed qRT-PCR analyses and found three out of
the four genes are truly differentially expressed between wild-
type and BORIS?/?testis (Table 2). 1700019B21Rik is a gene
with no known function that has been identified in mice and
rats but not in humans. TSP50 is a testis-specific protease
which is known to be a cancer testis antigen (47, 59). The
expression is regulated in part by methylation of the promoter
(22), is negatively regulated by p53 (56), and is abnormally
activated in a high proportion of breast cancers (59). The third
gene, Gal3st1, which encodes CST, showed the greatest reduc-
tion in transcripts in BORIS?/?testis (Table 2). Importantly,
CST?/?mice exhibit male infertility due to an arrest of sper-
matogenesis prior to the metaphase of the first meiosis (18),
paralleling the defect identified in BORIS?/?mice.
There are eight splice variants of CST expressed in different
tissues (16), but only one, form F, is expressed in testis (Fig. 4A
and B). To confirm the microarray analyses, we used qRT-
PCR to quantify total CST transcript levels and showed that
CST transcript levels were reduced more than 10-fold in testis
of BORIS?/?compared to wild-type mice at P14 (Fig. 4C). A
TABLE 1. Fertility analysisa
(female ? male) (n)
No. of litters
(mean ? SD)
(mean ? SD)
B6 ? BORIS?/?(4)
B6 ? BORIS?/?(4)
BORIS?/?? B6 (3)
BORIS?/?? B6 (6)
2.75 ? 0.50
2.00 ? 0.82
3.67 ? 0.58
3.17 ? 0.41
6.90 ? 3.48
8.38 ? 0.74
8.45 ? 2.16
9.74 ? 2.62
aTwo- to 3-month-old BORIS?/?or BORIS?/?mice were mated with 2- to
3-month-old B6 mice for 3 months, and mean litter numbers and litter sizes were
analyzed. All breeders produced offspring, and no significant difference was
found in litter number and litter size between BORIS?/?and BORIS?/?breed-
FIG. 2. Expression of GFP was analyzed by RT-PCR. cDNAs pre-
pared from each tissue are indicated: Te, testis; Br, brain; Li, liver; St,
stomach; Si, small intestine; Kd, kidney; P, positive control.
2476 SUZUKI ET AL.MOL. CELL. BIOL.
comparable reduction of CST expression was also found in
testes of mature mice (Fig. 4C). Furthermore, quantitation of
transcripts specifically for CST form F showed comparable
levels of reduction (Fig. 4D). We also confirmed that there was
a comparable reduction in the expression of CST in BORIS?/?
(-neo) testis (Fig. 4E and F). Analysis of CST transcript levels
during testicular development showed an abrupt induction of
expression at P14, when growing numbers of testicular cells
differentiated into spermatocytes, with increased levels of CST
transcripts being sustained thereafter (Fig. 4G). If BORIS reg-
ulates the expression of CST in testis, then BORIS should be
expressed at or before the time that CST is induced. Indeed,
BORIS transcripts were detectable from P2 and peaked at P14,
with expression sustained at relatively high levels thereafter,
consistent with the expression pattern of CST (Fig. 4H). Fur-
thermore, we found that CST was highly expressed in round
spermatids in correlation with the high expression of BORIS,
while there was no correlation between the levels of expression
of CST and CTCF (Fig. 4I to L). These results demonstrated
that expression of CST is reduced in testes of BORIS?/?mice
and suggest that BORIS might be directly involved in its tran-
FIG. 3. (A) Hematoxylin and eosin staining of testes prepared from P28 wild-type, BORIS?/?, and BORIS?/?(-neo) mice. Arrows indicate
multinucleated cells. Bars, 1 mm (left column); 100 ?m (right column). (B) Sizes of wild-type (blue) and BORIS?/?(red) testes at various stages. ?, P ?
0.05; ??, P ? 0.005. (C) TUNEL staining of testes from P28 wild-type (upper panel) and BORIS?/?(lower panel) mice. Bar, 100 ?m. TUNEL-positive
cells in each field were counted in testes of wild-type (blue) and BORIS?/?(red) mice. (D) Representative results of DNA flow cytometry performed
is shown in the inset. At least three mice were analyzed for each analysis. (E) Spermatocytes of wild-type (upper panel) and BORIS?/?(middle panel)
mice were stained with SCP3 (green), ?H2AX (red), and DAPI (blue). A dotted line indicates a boundary of adjacent cells. The lower panel shows a
high-magnification image of aberrantly accumulated SCP3 in BORIS?/?spermatocytes.
TABLE 2. Differentially expressed genes in P14 BORIS?/?testis
Fold changeabased on:
acDNAs prepared from P14 mice testes were used for qRT-PCR. qRT-PCR
analysis of Gal3st1 showed a larger difference than microarray results, probably
because qRT-PCR analysis has a greater dynamic range and returns more ac-
curate data than microarrays.
VOL. 30, 2010 BORIS REGULATES SPERMATOGENESIS THROUGH CST2477
FIG. 4. (A) Genomic structure of the CST gene. Open and filled boxes represent UTRs and open reading frames, respectively. It has been
reported that CST has eight splicing variants, all of which have the same coding region with different 5?-UTR sequences (16). CST form FTSis the
testis-specific form among splicing variants expressed in other tissues. Arrows denote the positions of primers used to evaluate the expression of
CST form FTotal(F) and total CST (T). Red arrows indicate the transcription start site of CST form FSSand CST form FTS. A dotted line indicates
sequences shown in panel B. (B) Sequences surrounding exon 1f. The gray box represents the reported exon 1f sequences. The numbering is
relative to the transcription start site of CST form FTS, which is marked by a red arrow. The reported transcription start site is marked by a dotted
red arrow. The red box shows BORIS/CTCF-contacting residues as determined in a methylation interference assay. Black arrows indicate primers
for CST form FSS. The dotted black arrow indicates a forward primer for CST form FTotal. DNA fragments used for EMSA are denoted by solid
lines or dotted lines in black or red. (C) cDNAs prepared from wild-type or BORIS?/?mouse testis at P14 (n ? 3) or at 3 months (n ? 3) were
2478SUZUKI ET AL.MOL. CELL. BIOL.
BORIS regulates expression of the testis-specific CST splic-
ing variant CST form FTS. To analyze the expression pattern of
CST, we performed qRT-PCR using cDNA prepared from
various tissues. As reported previously, CST was widely ex-
pressed in multiple tissues including testis although expression
of CST form F was restricted to stomach and testis (16) (Fig.
5A and B). Consistent with the known expression pattern of
BORIS, expression of CST and CST form F was greatly dimin-
subjected to qPCR to evaluate the expression of CST. Primers for total CST were used for the experiment. Expression levels are shown as the ratio
to wild-type level. Asterisks denote statistical significance (P ? 0.005). (D) cDNAs prepared from wild-type or BORIS?/?mouse testes at P14 (n ?
3) or at 3 months (n ? 3) of age were subjected to qPCR to evaluate the expression of CST form F. Primers for CST form FTotalwere used for
the experiment. Expression levels are shown as the ratio to the wild-type level. Asterisks denote statistical significance (P ? 0.005). (E) cDNAs
prepared from wild-type or BORIS?/?(-neo) mouse testis at P28 were subjected to qPCR to evaluate the expression of CST form F (n ? 3).
Primers for total CST were used for the experiment. Expression levels are shown as the ratio to wild type. The asterisk denotes statistical
significance (P ? 0.005). (F) cDNAs prepared from wild-type or BORIS?/?(-neo) mouse testis at P28 were subjected to qPCR to evaluate the
expression of CST (n ? 3). Primers for CST form FTotalwere used for the experiment. Expression levels are shown as the ratio to wild type. The
asterisk denotes statistical significance (P ? 0.005). (G) cDNAs prepared from wild-type testes were subjected to qPCR analysis to evaluate
the expression of CST during testis development. Primers for total CST were used for the experiment (n ? 3). Expression levels are shown as the
ratio to the P2 testis level. The asterisk denotes statistical significance (P ? 0.005). (H) cDNAs prepared from wild-type testes were subjected to
qPCR analysis to evaluate the expression of BORIS during testis development (n ? 3). Expression levels are shown as the ratio to the P2 testis
level. (I) Expression levels of BORIS, CST, and CTCF were analyzed by RT-PCR. cDNAs were prepared from liver (Li), spermatocytes (SC), and
round spermatids (RS). (J to L) cDNAs prepared from SC and RS were subjected to qPCR analysis to evaluate the expression levels of BORIS
(J), CST (K), and CTCF (L) (n ? 3). Expression levels are shown as the ratio to the level in spermatocytes. Asterisks denote statistical significance
(P ? 0.05).
FIG. 5. (A and B) Expression of CST and CST form F in various tissues of wild-type and BORIS?/?mice was analyzed by qPCR (n ? 3).
Primers for CST form FTotalwere used in panel B. Expression levels are shown as the ratio to wild-type testis levels. Asterisks denote statistical
significance (P ? 0.005). (C) BORIS binding to the promoter region of exon 1f was analyzed by EMSA using fragments shown in Fig. 4B.
Luciferase protein was used as a negative control. Luc, luciferase; B, BORIS. (D) Methylation interference assay of the BS2 fragment using 11-ZF.
Partial sequences of the fragment are shown. Only the bottom strand is shown, as the top strand did not show any differences. Left lane, unbound
fragments; right lane, bound fragments. Asterisks indicate contacting guanine residues. (E) Expression of CST form FSSin stomach and testis of
wild-type and BORIS?/?mice was analyzed by qPCR (n ? 3). Expression levels are shown as the ratio to wild-type testis. Asterisks denote statistical
significance versus the testis sample (P ? 0.005). (F) The ratio of CST form FSSin CST form FTotalwas analyzed by qPCR (n ? 3). Due to the
massive and specific reduction of CST form FTSexpression in BORIS?/?testis, the ratio of CST form FSSin CST form FTotalwas slightly increased
in BORIS?/?testis. Asterisks denote statistical significance versus the testis sample (P ? 0.01). (G) Methylation status around exon 1f was analyzed
by bisulfite sequencing. Open and filled circles represent unmethylated and methylated cytosines, respectively. (H) Activity of the CST form FTS
promoter was analyzed by luciferase assay (n ? 3). Empty vector (vec), BORIS, or CTCF expression vectors were cotransfected with each
construct. Numbers indicate the 5? end of each construct on the CST form FTSpromoter region. Luciferase activities are shown as the ratio to the
empty vector-transfected sample. Asterisks denote statistical significance versus empty vector-transfected sample (P ? 0.05).
VOL. 30, 2010BORIS REGULATES SPERMATOGENESIS THROUGH CST2479
ished in BORIS?/?testis but not in the other tissues, including
stomach, suggesting that CST form F promoter contains a
potential BORIS binding site (Fig. 5A and B).
To determine if BORIS would indeed bind to the promoter
region, we performed EMSAs using fragments derived from
the promoter region of CST form F (Fig. 4B) and found that
BORIS bound to fragment BS2 (Fig. 5C). This fragment was
also capable of binding CTCF (Fig. 6C). Neither BORIS nor
CTCF (data not shown) bound any of the other fragments
tested. To identify the BORIS binding site in fragment BS2, we
performed methylation interference assays of the fragment
using the 11-ZF domain of CTCF, based on its known strong
binding to CTCF target sequences in vitro and the shared
recognition sequence of BORIS and CTCF (Fig. 5D). We
found that 11-ZF bound to tandem guanine residues that are
located within exon 1f (Fig. 4B).
These results indicate that a BORIS/CTCF target site is
located inside exon 1f. Although it was reported that BORIS/
CTCF target sites could exist inside exonic sequences (52),
most proteins that activate transcription bind upstream of the
transcription start site. In view of the finding that the mecha-
nisms governing activation of the CST form F promoter
seemed to differ between testis and stomach (Fig. 5B), we
hypothesized that there is a testis-specific transcription start
site downstream of the BORIS/CTCF target site different from
that previously described for exon 1f. To analyze this possibil-
ity, we performed 5?-RACE using testis RNA and found that
all 10 CST transcripts analyzed started from exon 1f down-
stream from the BORIS/CTCF target site (Fig. 4B). Further-
more, we confirmed that CST form F transcripts highly ex-
pressed in stomach were initiated upstream from the BORIS/
CTCF target site (Fig. 5E). Virtually all the CST form F
transcripts expressed in testis were the short form, while the
CST form F expressed in stomach was exclusively the longer
form (Fig. 5F). Hereafter, we call the short form of the CST
form F transcript CST form FTS, for testis specific, the origi-
FIG. 6. (A) Sequences of wild-type and mutants of the CST form FTSpromoter. Asterisks indicate contacting guanine residues. Four contacting
guanines were converted into adenines (arrowheads) in mutant constructs. Numbering shows the positions relative to the transcription start site
of CST form FTS. (B) Binding of BORIS to the mutated BS2 fragment was analyzed by EMSA. Luc, luciferase; B, BORIS. (C) Binding of 11-ZF
and CTCF to the mutated BS2 fragment was analyzed by EMSA as for panel B. C, CTCF. (D) Luciferase assays were performed using a mutated
promoter of the CST form FTS(n ? 3). Empty vector or BORIS expression vector were cotransfected with each construct. Luciferase activities
are shown as the ratio to empty vector-transfected sample. The asterisk denotes statistical significance (P ? 0.05). Vec, empty vector; B, BORIS.
(E) Effect of BORIS on CST form FTotalexpression was analyzed by transient transfection of a BORIS expression vector into NIH 3T3 cells.
Expression of CST form FTotalwas analyzed by qPCR (n ? 3). Expression levels are shown as the ratio to mock-transfected sample. The asterisk
denotes statistical significance versus empty vector-transfected sample (P ? 0.005). Vec, empty vector. (F) Binding of BORIS on the CST form
FTSpromoter (P) and 1.6 kb upstream of the transcription start site (5?) in testis was analyzed in a ChIP-qPCR assay using anti-BORIS antibody
(n ? 4). The asterisk denotes statistical significance (P ? 0.05). (G) Recognition of BORIS/DNA complexes by BORIS antibody was validated by
supershift assay. Fragment BS2 was preincubated with BORIS, CTCF, or luciferase followed by incubation with antibody against BORIS or CTCF.
The arrow indicates the shifted band. Arrowhead 1 indicates a supershifted band with BORIS antibody, and arrowhead 2 indicates a supershifted
band with CTCF antibody. B, BORIS; L, luciferase; C, CTCF. (H) Effects of BORIS and 5-azadC on CST form FTotalexpression were analyzed
by transient transfection of a expression vector into NIH 3T3 cells either treated or not with 1 ?M 5-azadC. Expression of CST form FTotalwas
analyzed by qPCR (n ? 3). Cells were cultured with or without 5-azadC for 24 h followed by transient transfection. Cells were further cultured
for 48 h and harvested for analysis. Expression levels are shown as the ratio to empty vector-transfected cells without 5-azadC.*, statistically
significant versus empty vector-transfected cells without 5-azadC (P ? 0.01);**, statistically significant versus empty vector-transfected cells with
5-azadC (P ? 0.005). E, empty vector; B, BORIS; C, CTCF.
2480SUZUKI ET AL.MOL. CELL. BIOL.
nally reported longer form CST form FSS, for stomach specific,
and both forms when measured together as CST form FTotal.
Methylation of CpG sequences in promoter regions is one of
the major mechanisms responsible for suppression of gene
expression. To analyze the methylation status of the CST form
FTSpromoter, we performed bisulfite sequencing and found
that the region was highly methylated in tissues that didn’t
express CST form FTS—kidney, brain, and stomach—but was
fully unmethylated in spermatocytes, even in BORIS?/?mice
(Fig. 5G). This suggested that promoter methylation was re-
sponsible for suppression of CST form FTSexpression, al-
though BORIS being present or absent had no effect on the
methylation status of its promoter.
Taken together, these results indicate that transcription of
CST form FTS, which is initiated downstream from the BORIS/
CTCF target site, is regulated by BORIS in testis, while tran-
scription of CST form FSSis BORIS independent.
BORIS activates CST expression through binding to the
BORIS/CTCF target site. To further understand the role of
BORIS in regulating the CST form FTSpromoter, we per-
formed luciferase assays using different fragments from the
promoter region (Fig. 5H). All constructs cotransfected with a
BORIS expression vector showed activation of the promoter,
while cotransfection with a CTCF expression vector had no
appreciable effect. This result suggests that BORIS activates
the CST form FTSpromoter by binding to the BORIS/CTCF
target site while CTCF cannot.
As another approach to establishing that activation of the
CST form FTSpromoter was due to binding of BORIS to the
BORIS/CTCF target site, we performed EMSA using frag-
ments mutated on the residues recognized by the 11-ZF (Fig.
6A). BORIS did not induce a mobility shift with the mutated
BS2 fragments, a finding common to the 11-ZF and full-length
CTCF, indicating that BORIS as well as CTCF bound to the
sequence (Fig. 6B and C). Furthermore, BORIS-induced ac-
tivation of the promoter was diminished when the same mu-
tation was introduced in the construct used for luciferase as-
says (Fig. 6D). We also confirmed that exogenous expression
of BORIS, but not CTCF, induced expression of CST form
FTotalin NIH 3T3 cells (Fig. 6E). To analyze binding of BORIS
to the CST form FTSpromoter region, we performed a ChIP-
qPCR assay using whole testes derived from wild-type and
BORIS?/?mice. Consistent with the in vitro findings, we found
specific enrichment of BORIS on the CST form FTSpromoter
region from wild-type testis but not from BORIS?/?testis (Fig.
6F). The validity of using the rabbit anti-mouse BORIS anti-
bodies for ChIP was confirmed by supershift assays (Fig. 6G).
Taken together, these results indicate that BORIS activates
expression of CST form FTSthrough binding to the BORIS/
CTCF target site.
To understand the physiological roles played by BORIS, we
generated BORIS knockout mice and found that BORIS is
critical to normal spermatogenesis (Fig. 3). Although there
were no apparent abnormalities in the population of testicular
cells at P14, we found delayed spermatogenesis as early as P21,
suggesting that BORIS functions in meiosis. We conclude that
the reduced size and the hypocellularity of testes in BORIS?/?
mice are probably due to a failure of meiosis resulting in the
apoptotic death found in BORIS?/?testis.
Using microarray analyses, we found that BORIS regulated
the expression of CST, as demonstrated by the greatly reduced
levels of transcripts in BORIS?/?testis (Table 2). This alter-
ation in CST expression was found in the testis but not the
somatic tissues of BORIS?/?mice, keeping with the testis-
specific expression of BORIS (Fig. 5A and B). The defect in
spermatogenesis seen in BORIS-deficient mice at meiosis is a
near replica of that seen in testes of CST knockout mice (18).
On the other hand, although CST knockout mice showed com-
plete sterility, BORIS knockout mice were still fertile. Since
production of sperm far exceeds the number necessary for
fertility, it is likely that residual expression of CST found in
BORIS?/?testis is sufficient to maintain fertility even though it
causes significant defect in spermatogenesis.
We found that CST was expressed in testis as a novel testis-
specific isoform, CST form FTS, which has shorter exon 1f
sequences (exon 1fS) than described previously (16). CST form
FTSwas the dominant form of CST expressed in testis even in
BORIS KO mice (Fig. 5E and F), indicating that BORIS is
important for activation of the CST form FTSpromoter but not
for defining the transcription start site of that form. Consistent
with the testis-specific expression of CST form FTS, the pro-
moter for this isoform was highly methylated in somatic tissues,
including stomach, which expressed CST form FSS, while it was
unmethylated in spermatocytes (Fig. 5G). This suggests that
methylation of the promoter is responsible for the repressing
expression of CST form FTS. The promoter for this isoform
was unmethylated in spermatocytes not only in wild-type mice
but also in BORIS?/?mice. The unmethylated status of the
promoter may be responsible for the incomplete repression of
CST expression in BORIS?/?testis, while also indicating that
BORIS is not responsible for demethylation of the promoter.
We confirmed that ectopic expression of BORIS in somatic
cells treated with 5-aza-2?-deoxycytidine (5-azadC), which in-
duces demethylation of CpG dinucleotides, synergistically ac-
tivates the expression of CST form FTS(Fig. 6H), although
ectopic expression of BORIS alone or treatment with 5-azadC
alone could also induce expression. Taken together, these re-
sults suggest that demethylation of the promoter is a prereq-
uisite for BORIS to fully activate CST form FTSexpression.
We showed that BORIS binds the CST form FTSpromoter.
Methylation interference assays followed by EMSA with mu-
tated fragments revealed that the exact BORIS binding site in
the promoter is highly homologous to the TAGGGGG-con-
taining CTCF/BORIS binding sequence mapped by DeJong
and coworkers in the testis-specific ALF gene promoter (8, 26),
which has been termed the consensus CTCF binding site as
identified by genome-wide ChIP analysis (27). Consistent with
the finding of a BORIS/CTCF target site in the promoter,
ectopic expression of BORIS activated the CST form FTSpro-
moter, but not the promoter mutated in the BORIS/CTCF
target site. It is interesting that both BORIS and CTCF can
recognize the BORIS/CTCF target site, but only BORIS can
activate the promoter. The expression pattern of BORIS,
CTCF, and CST in spermatogenic cells also suggests a BORIS-
specific function in regulation of CST expression. These data
provide a clear demonstration that BORIS is functionally dif-
ferent from CTCF, even though they have the same recogni-
VOL. 30, 2010BORIS REGULATES SPERMATOGENESIS THROUGH CST 2481
tion sequences. Since the amino and carboxy termini of BORIS
have no homology with the parallel sequences in CTCF, the
distinct molecular functions of BORIS may be attributable to
the unique protein-protein interactions specified by these se-
quences. Our observations on the role of BORIS binding site
in activation of the CST form FTStranscription are remarkably
consistent with results on regulation of testis-specific TFIIA
?/?-like factor (ALF) gene promoter obtained by DeJong and
coworkers, who showed that unlike CTCF, expression of
BORIS had an activating effect on the ALF promoter-reporter
construct (9, 26). However, ALF only starts to express at the
time point we performed microarray analysis, and that may be
why we missed it as a target (15).
Although orthologs of CST can be found in species from
zebrafish to humans, the sequence of exon 1fSis found only in
mammals. This suggests that the testis-specific regulation of
CST expression by BORIS evolved after mammalians di-
verged. Interestingly, the sequence of exon 1fSis not well
conserved among mammals, while the BORIS/CTCF target
site as well as the splice site flanking exon 1fSare highly
conserved (see Fig. S3 in the supplemental material). This
suggests that the regulation of CST form FTSexpression by
BORIS, which depends on the BORIS/CTCF site, is important
in mammalian species, although the 5?-UTR sequence of exon
1fSitself is not. It is noteworthy that BORIS is expressed in a
variety of tissues in lower vertebrates, while expression is re-
stricted to the testis in mammals (20). This suggests that the
acquisition of BORIS-specific regulatory mechanisms devel-
oped in conjunction with the functional specialization of
BORIS to testicular germ cells.
Aside from CST, we found a few other genes that are dif-
ferentially expressed in testes of wild-type and BORIS-defi-
cient mice. This is probably due in part to the fact that we
analyzed testes from P14 mice to identify BORIS target genes
that were responsible for initiating the spermatogenesis defect.
This is the earliest stage of testicular development at which we
can expect to identify differentially expressed genes. The fact
that spermatogenesis is disturbed in BORIS KO mice makes
the identification of potential BORIS target genes that func-
tion later in spermatogenesis difficult. Transcriptional profiles
of purified testicular cell subsets would be certain to yield
additional targets. However, we also found several genomic
targets for BORIS binding that could not be correlated with
transcriptional regulation, suggesting other BORIS functions.
Further studies are required to examine these possibilities.
Although we found that BORIS is a major determinant of
normal spermatogenesis, BORIS?/?male mice were still fer-
tile, indicating that there are mechanisms that can compensate
for the loss of BORIS. Since CTCF is the only known paralog
for BORIS, CTCF may partially compensate for the loss of
BORIS, enabling BORIS?/?male mice to remain fertile. To
determine if this model is correct, it would be necessary to
analyze BORIS/CTCF double mutant mice. In addition, we
cannot rule out the possibility that there is a protein other than
CTCF that can compensate for the loss of BORIS. Finally, the
phenotypes associated with different gene knockout mice are
notoriously strain dependent (1, 45). The mice evaluated in the
current study had a mixed 129/B6 genetic background such
that progressive introgression onto a pure background such as
B6 could result in a strain with significant phenotypic differ-
Based on previous studies of BORIS and CTCF and their
abilities to recognize common target sites, it is likely that
BORIS will be found to have functions other than regulating
spermatogenesis. Methylation-sensitive binding of CTCF to
ICRs of H19/Igf2 (24), Rasgrf1 (58), and Kcnq1 (13) regulates
allele-specific gene expression of these imprinted loci in mouse
and human somatic cells. However, ICRs alone are neither
necessary nor sufficient for resetting of imprinting upon germ
line transmission (32). Additional ICR-targeting sequences
have been mapped for the Rasgrf1 ICR (58) but remain un-
known for H19/Igf2 and other ICRs. Therefore, remethylation
may indeed occur upon replacement of BORIS to CTCF as
suggested (37) and be targeted to paternal ICRs from a distant
BORIS binding site that remains to be mapped in vivo (32).
Although offspring of BORIS?/?mice develop normally, indi-
cating that at least some sperm can develop normally without
BORIS, there might be defective germ cells with aberrant im-
printing destined to die in BORIS?/?testis, a suggestion con-
sistent with the high rate of apoptosis seen in testes of BORIS-
Most promising, perhaps, is the finding that expression of
Tsp50 is markedly altered in BORIS-deficient testes. TSP50 is
known as a CTA (47, 59), is overexpressed in a high proportion
of breast cancers (59), is a gene with promoter methylation
associated with expression (22), and has a near-canonical
CTCF target sequence in the promoter (data not shown). Fur-
ther studies of this gene, its regulation, and its importance as a
CTA may link it functionally to BORIS regulation of CTA and
to BORIS as a CTA target for cancer treatment (36).
This research was supported by the Intramural Research Program of
the NIAID, NIH.
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