A 34 bp fragment located at191 to 158 from a translation start posi-tion, and containing the TCCCCTCCC sequence, was involved in enhan-cing promoter activity, which was assessed by lucif
Trang 1promoter function
Hiroshi Suganuma1, Maki Kumada1, Toshinori Omi1, Takaya Gotoh1, Munkhtulga Lkhagvasuren1, Hiroshi Okuda1,2, Toyomi Kamesaki1,2, Eiji Kajii1,2and Sadahiko Iwamoto1
1 Division of Human Genetics, Center for Community Medicine, Jichi Medical School, Japan
2 Division of Community Medicine, Center for Community Medicine, Jichi Medical School, Japan
The rhesus (Rh) blood group antigens are of
consider-able importance in transfusion medicine as well as in
newborn or autoimmune hemolytic diseases due to their
high antigenicity [1] The Rh antigens are carried by two
distinct but homologous integral membrane proteins of
30–32 kDa, which have been isolated by
immunopreci-pitation using anti-Rh antibodies [2] The corresponding
cDNAs, RHCE [3,4] and RHD [5–7] have been cloned
They differ in only 31–35 of 417 amino acid residues
and have been mapped in tandem on chromosome
1q34.3–36.1 Genomic organization of the RH locus has
revealed that RHD and RHCE face each other at their
3¢ tails, and that the gene SMP1 is interspersed between
them [8] Wagner et al identified two 9 kb
transposon-like DNA segments, called ‘rhesus boxes’ upstream and
downstream of the RHD gene They speculated that these boxes are involved in the regulation of SMP1 because they encode a GC-rich region at the 3¢ ends [9] However, whether rhesus boxes are involved in regula-tion of the RH locus remains unknown
Rh transcripts are distributed among cells of hema-topoietic lineage with erythroid features [3] The expression of RhD and CE antigens increases synergis-tically with the maturation of erythroblasts [10] The promoter sequences of the RH genes support erythroid specific expression of the Rh antigens [11] The human b-globin locus is composed of five tandem arrayed genes and it is controlled by a locus control region (LCR) localized between positions )21.5 and )6.1 kb from the e globin cap site [12,13] The LCR can open
Keywords
Rh blood group antigen; promoter;
transcription cofactor; Aly ⁄ REF
Correspondence
S Iwamoto, Division of Human Genetics,
Center for Community Medicine, Jichi
Medical School, 3311-1
Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498,
Japan
Fax: +81 285 44 49
Tel: +81 285 58 7342
E-mail: siwamoto@jichi.ac.jp
Note
Hiroshi Suganuma and Sadahiko Iwamoto
contributed equally to this work
(Received 26 November 2004, revised 17
March 2005, accepted 23 March 2005)
doi:10.1111/j.1742-4658.2005.04681.x
The rhesus (Rh) blood group antigens are of considerable importance in transfusion medicine as well as in newborn or autoimmune hemolytic diseases due to their high antigenicity We identified a major DNaseI hypersensitive site at the 5¢ flanking regions of both RHD and RHCE exon
1 A 34 bp fragment located at)191 to )158 from a translation start posi-tion, and containing the TCCCCTCCC sequence, was involved in enhan-cing promoter activity, which was assessed by luciferase reporter gene assay A biotin-labelled 34 bp probe isolated an mRNA transporter pro-tein, Aly⁄ REF The specific binding of Aly ⁄ REF to RH promoter in eryth-roid was confirmed by chromatin immunoprecipitation assay The silencing
of Aly⁄ REF by siRNA reduced not only the RH promoter activity of the reporter gene but also transcription from the native genome These facts provide second proof of Aly⁄ REF as a transcription coactivator, initially identified as a coactivator for the TCRa enhancer function Aly⁄ REF might be a novel transcription cofactor for erythroid-specific genes
Abbreviations
ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; HEL, human erythroleukemic cell line; HS, hypersensitive sites; LCR, locus control region.
Trang 2the b-globin locus, enhance transcription and control
the timing and choice of gene for transcription
within the locus [13] The LCR is characterized by the
presence of five DNaseI hypersensitive sites (HS) that
encode multiple DNA motifs for transcription factors
The present study identifies an enhancer element and a
protein that promotes its activity in the major HS of
both RH genes in an erythroleukemic cell line
Mapping the DNaseI HS of RH loci
We investigated the major regulatory region of RH
gene expression using in vivo DNaseI hypersensitivity
analysis in a human erythroleukemic cell line (HEL),
which expresses Rh antigens on the cell surface [14]
We prepared three probes with which to explore two
RHloci Probe 1 that encoded exon 1 and the 5¢ end of
intron 1 of the RHD and CE genes revealed 3.7 kb bands (solid arrow head in Fig 1) along with weakened
20 kb (CE gene) and 18 kb (D gene) bands Probes 8 and 10 revealed weak bands (grey arrowheads), indica-ting control elements in intron 9 Probes encoding the unique sequence in the rhesus box did not reveal any apparent DNaseI hypersensitivity (data not shown) Thus, the rhesus boxes are not the major controlling region of the RH loci, but the 5¢ flanking sequences
of RHCE and RHD exon 1 were the most important regulatory region studied
Functional mapping of regulatory regions
in RHD upstream sequence The )12 159 bp sequence from the translation start site of the RHD gene was inserted into the pGL3
Fig 1 DNaseI hypersensitivity mapping of the RH gene in HEL cells Probes are indicated in the locus map Restriction sites of EcoRV and exons of each gene are shown by vertical lines on the horizontal line and gray lines across it, respectively DNaseI concentrations increase from 0 to 15 lgÆmL)1(triangles) Extracted DNA was digested by EcoRV Southern blots show positions of major (arrowheads) and minor (grey arrowheads) HS.
Trang 3B
Fig 2 Identification of the functional element by a functional assay of the 5¢ flanking region of the human RH gene (A) Top, repetitive sequence map The full-length construct, pGL-RH-12159, is the parental vector to induce deletion derivatives Constructs were transfected into HEL cells and relative light units were measured (B) The pGL-RH-238 construct induced further deletions (dashed line) or mutations (grey letters) according to PCR Relative light units are shown as percentage values of those of parental vector, pGL-RH-238 Rectangles, GATA motifs Oligo probes used for electrophoretic mobility shift assay are indicated by horizontal bars with numerals.
Fig 3 Purification of DNA binding protein to probe 2 (A) EMSA study of the promoter sequence of the human RH gene Four double-stranded oligonucleotide probes prepared as shown in Fig 2B were incubated with nuclear extracts of HEL cells Competition proceeded with cold competitors indicated above Numbers with M indicate mutant competitors The supershift assay included the anti-(GATA-1) mAb (B) SDS ⁄ PAGE of purified by EMSA probe 2 Samples from each purification cycle were analysed by 5–15% gradient SDS ⁄ PAGE and silver staining Lane 1, nuclear extract; lanes 2 and 3, 1 ⁄ 10 aliquot of first and second cycle products with mutant probe; lanes 4 and 5, 1 ⁄ 10 aliquot of first and second cycle products with wild-type probe Left, marker proteins Bold letters indicate three internal amino sequences
of trypsin-digested fragments of protein band in sequence nominated by MASCOT analysis (C) ChIP assay in HEL and HeLa cells The chroma-tin enriched by negative control antibody (NegCon), anti-TFIIB or anti-Aly Ig and whole chromachroma-tin (imput) were amplified by PCR primers indicated on the left.
Trang 4B
C
Trang 5vector Serial deletion constructs from this parental
vector revealed that a DNA fragment from )238 to
)140 was closely involved in transcriptional activity
and that a further upstream sequence acts as a
suppressor in HEL cells (Fig 2A) Deletion and
site-directed mutants of the )238 construct
(pGL3-RH-238) showed that the absence of a 34 bp fragment
from )191 to )158 (pGL3-RH-238del) decreased the
activity to less than 20% of the original (Fig 2B)
Dis-ruption of the GATA motifs, especially the proximal
one, decreased the activity to 20%
(pGL3-RH-238Mut3) and a mutation in the C-rich region within
the 34 bp sequence decreased the activity by 50%
(pGL3-RH-238Mut1) These data suggested that the
DNA fragment from )191 to )158, especially the
C-rich sequence, is involved in the enhancer activity
Identification of the protein that binds to the
putative regulatory region
Three overlapping double-stranded oligo probes
exam-ined DNA binding proteins at the 34 bp sequence
Among the probes, the shift bands in the
electrophore-tic mobility shift assay (EMSA) determined by probe 2
were the most significant (Fig 3A) The shift bands
were partially competed out by a 50-fold excess of
wild-type competitor but not by a mutant probe (2 m)
or by either of the neighbouring probes 1 and 3,
sug-gesting that TCCCCTCCC sequence unique for probe
2 was the motif for the transcription factor
Anti-EKLF and anti-Sp1 antibody did not influence the shift
band mobility, while the binding motif of EKLF
(CCCACCC) and Sp1 (CCCGCCCC) resembled the
C-rich sequence (data not shown) Probe 4 encoding
the proximal GATA motif showed an intense band that
was super-shifted by the anti-GATA1 antibody,
indica-ting the GATA1 is closely involved in the function of
this promoter
We used biotin-labelled double-stranded probes to
isolate the protein that bound to probe 2 While the
mutant probes did not retain any protein after the
sec-ond wash, the wild-type probe bound a 35-kDa protein
(Fig 3B) A mass spectrogram of trypsin-digested
pep-tides of the band and the internal peptide sequences
revealed that the protein recovered by the wild-type
probe was Aly⁄ REF
The binding of Aly⁄ REF to the RH promoter was
verified by chromatin immunoprecipitation (ChIP)
assay Enrichment of a DNA fragment encoding the
RH promoter was observed by anti-ALY antibody in
two independent experiments using HEL cells, while
the RH promoter was not condensed from chromatin
of nonerythroid HeLa cells (Fig 3C)
Silencing of Aly⁄ REF decreased RH promoter activity
To determine whether Aly⁄ REF actually activates the
RH promoter, the reporter plasmids pGL3-RH-238 and )158 were induced into HEL cells with a plasmid expressing Aly⁄ REF siRNAs The Aly ⁄ REF siRNA encoding nt 283–303 (pSilencerALY283-303) signifi-cantly (P < 0.05) decreased the RH promoter activity
of the pGL3-RH-238 construct but the decrease was not significant when pGL3-RH-158 was the reporter (Fig 4A) In contrast to the RH promoter, SV40 pro-moter activity was enhanced by the siRNAs These results indicated that the C-rich region in the 34 bp sequence is specific for Aly⁄ REF or for DNA binding proteins associated with Aly⁄ REF
Quantitative RT-PCR showed that Aly⁄ REF siRNA decreased the amount of Aly⁄ REF mRNA (Fig 4B) The mRNA reduction by pSilencerALY285-303 was greater than that induced by pSilencerALY45-65 The amount of Rh transcripts were significantly decreased when pSilencerALY285-303 was induced in HEL cells, although the decrease induced by pSilencerALY45-65 was not significant, which might reflect its lower efficiency of reduction for Aly⁄ REF mRNA The Ct value of GAPDH mRNA was not influenced by the induction of Aly⁄ REF siRNA (data not shown) Thus, the decreased Rh mRNA and luciferase activity did not result from a dysfunction in mRNA transportation but from a downregulation of RH promoter function caused by a decrease of Aly⁄ REF protein
Discussion
We identified a DNaseI HS region in the 5¢ flanking sequence of exon 1 of both RHCE and RHD, which appeared to be the major regulatory region within RH loci Neither in vivo DNaseI hypersensitivity analysis nor luciferase assays suggested that loci control regions are located in rhesus boxes In the 5¢ flanking sequence
of RH exon 1, the C-rich sequence acts as an enhancer element, whereas the GATA element was the most important motif in the same manner as other eryth-roid-specific genes
We isolated Aly⁄ REF protein using double-stranded oligo probes encoding the C-rich sequence The binding
of Aly⁄ REF at the RH promoter in HEL cells was con-firmed by ChIP assay Its specific binding to the RH pro-moter in erythroid cells was shown by its background level accumulation from chromatin of the cervical cancer cell line, HeLa, which does not express Rh antigen Aly⁄ REF has two RNA binding domains and is involved in RNA transportation from the nucleus
Trang 6[15,16], but it was initially identified as a coactivator
of LEF-1 and AML-1 for TCRa enhancer function
[17] Bruhn et al have shown that Aly⁄ REF itself has
no specific DNA or RNA sequence motif and
estima-ted that it acts as a context dependent coactivator
However, they also remarked that the ternary
com-plex of Aly⁄ REF with LEF-1 and DNA was
depend-ent on the flanking sequence of the LEF binding
motif, suggesting that Aly⁄ REF also weakly interacts
with the sequences [17] Our data suggested that
Aly⁄ REF interacts with a preferential sequence The
mutation in TCCCCTCCC reduced the promoter
activity by 50% (Fig 2B), as it lost the ability to
compete out the shift band in EMSA (Fig 3A) and
the mutant biotin labelled probe failed to retain
Aly⁄ REF protein (Fig 3B) Another explanation for these phenomena is that an unknown factor interacts with the TCCCCTCCC motif and that an overwhelm-ing molar excess of Aly⁄ REF protein interacts with it
as a scaffold These notions remain to be investi-gated
The knockdown experiment with Aly⁄ REF siRNA
in HEL cells reduced the RH promoter activity of both the reporter plasmid and the native genome The reduction was indeed subtle, which might result from low efficiency of pSilencer transfection into HEL cells, and from partial magnitude of Aly⁄ REF activity on the promoter as shown by mutageneis analysis at the motif (Fig 2B) In contrast to the RH promoter, the reduction in Aly⁄ REF significantly enhanced SV40
A
B
Fig 4 Interference of siRNA in Aly ⁄ REF.
(A) HEL cells were transfected with
pSilenc-er harbouring a random control sequence,
ALY45-65 or ALY285-303 and reporter pGL3
inserted with RH-158, RH-238 or the SV40
promoter indicated below and internal
con-trol pRL-CMV vector Relative luciferase
assays were performed 24 h later Relative
luciferase activities normalized by
pSilencer-control in each reporter vector are shown
with mean (bars) and SD (lines) values of
triplicated transfection experiments (B) HEL
cells were transfected solely with pSilencer
constructs indicated below and Aly⁄ REF or
Rh mRNA levels were assessed by
quantita-tive RT-PCR Relaquantita-tive mRNA amounts
nor-malized by pSilencer-control are shown with
SD values of triplicated transfection
experi-ments Asterisks indicate statistical
signifi-cance: *P < 0.05 and **P < 0.01.
Trang 7promoter activity Bruhn et al have also shown the
promoter specific enhancement by Aly⁄ REF; ALY
antisense oligonucleotide decreased TCRa promoter
function, but did not affect Rous sarcoma virus
promoter [17] These data suggested that Aly⁄ REF
protein is actually involved in the expression of Rh
protein depending on the promoter sequence
In the same way as glycophorin A and B [18], two
RH genes are synergistically expressed along with
erythroid maturation and they might be under the
con-trol of individual enhancers in each gene The C-rich
region with which Aly⁄ REF interacts is a control
element for both RH loci Various transcription factors
that interact with erythroid specific gene promoters
or enhancers have been identified GATA-1, GATA-2,
NF-E2, EKLF, FOG, LMO, SCL and Lbd have been
characterized as transcription factors of erythroid
specific genes [19,20] and Aly⁄ REF might be a novel
addition to this group
Rh antigens, especially RhD, cause harmful
hemo-lysis in neonates and during transfusion medicine [1]
Understanding the regulatory mechanisms of Rh
anti-gen expression will help to avoid harmful hemolytic
reactions in newborns or in auto-immune hemolytic
diseases
Experimental procedures
DNaseI hypersensitivity analysis
HEL cells obtained from the Health Science Research
Resources Bank (Osaka, Japan) was maintained in
RPMI1640 containing 10% (v⁄ v) fetal bovine serum
Nuclei from HEL cells were isolated as described [21]
Aliquots (0.25 mL) of nuclei at a density of 2· 107ÆmL)1
were digested with increasing concentrations of DNaseI
(0–15 lgÆmL)1) and then DNA was extracted as
des-cribed Purified DNA (10 lg) was digested with EcoRV,
resolved by electrophoresis on 0.8% agarose gels and
transferred onto Hybond-N+ nylon membranes
(Amer-sham Pharmacia Biotech, Little Chalfont,
Buckingham-shire, UK) The blots were initially hybridized with a
human RH exon 1 probe (Fig 1, probe 1) After
auto-radiography, the membranes were stripped and
succes-sively rehybridized with probes 8 and 10, encoding exons
8 and 10, respectively
Plasmid constructs and transduction and reporter
enzyme assay
The )12 159 bp sequence from the translation start site
of the RHD gene (GenBank accession no AB029152)
[22] that encodes almost 85% of the rhesus box was
cloned as described and inserted into the pGL3 vector Serial deletion derivatives were constructed from this par-ental vector using the restriction sites Site-directed muta-genesis or segmental deletion in the predicted motifs of the minimum promoter proceeded using the PCR-based method of Imai et al [23] as described For transfection,
5· 105
HEL cells were cotransfected with the pGL3-RH plasmid and pRL-CMV using LipofectAMINE (Invitro-gen, Carlsbad, CA) Twenty-four hours later, cells were harvested and relative light units (firefly⁄ Renilla light units) were measured using a Dual-Luciferase reporter assay system (Promega, Madison, WI, USA) and a TD-20⁄ 20 luminometer (Turner Designs, Sunnyvale, CA, USA) At least three independent transfection assays were performed
EMSA EMSA reactions were performed using nuclear extracts of HEL cells and [32P]dCTP-labelled double-stranded probes (Fig 2B) For supershift assays, 1 lL of rat mAb anti-GATA1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added Samples were incubated for 30 min at room temperature and resolved on 5% acrylamide gels in 0.5· TBE buffer at room temperature The gels were dried and exposed to X-ray film
Purification and identification of the protein that binds to the regulatory motif
The protein that bound to probe 2 in the EMSA was puri-fied using avidin-biotin as described [24] with some modifi-cation The biotinylated probe was composed of a chemically synthesized oligonucleotide with a biotin-phos-phoramidite tail (lower strand) and an unlabelled upper strand oligonucleotide as follows: biotin-5¢-GGGACTAT GATGATGGGGAGGGGAGGAAATGT-3¢ and 5¢-ACA TTTCCTCCCCTCCCCATCATAGTCCC-3¢ To exclude the possibility of nonspecific protein binding, we prepared the following mutant probe: biotin- 5¢-GGGACTATGAT GATGGGTTTGTGAGGAAATGT-3¢ and 5¢-ACATTTC CTCACAAACCCATCATAGTCCC-3¢ HEL cell nuclear proteins carried by the probes were separated using strept-avidin-magnetic beads (Qiagen, Valencia, CA, USA) Bead suspensions were washed and then trapped proteins were extracted in high-salt buffer until a 10% aliquot of the extracted protein resolved as a single protein band in SDS⁄ PAGE as shown by silver staining The protein band was excised and digested with trypsin The peptide fragments were examined by MALDI-TOF-MS and LC-MS⁄ MS performed at the ProPhoenix Institute (Hiro-shima, Japan) The mass fingerprint data were analysed by mascot and the internal amino acid sequences were com-pared with the NCBInr databases
Trang 8ChIP assay
HEL and HeLa cells were fixed for 10 min in RPMI1640
con-taining 1% (v⁄ v) formaldehyde The nuclei from the fixed
cells were sonicated in the presence of protease inhibitor
cocktail and the sheared chromatins were incubated with
mouse ALY Ig (ImmuQuest, Cleveland, UK),
anti-TFIIB or negative control IgG provided by Active Motif
(Carlsbad, CA, USA) Enrichment of chromatin fragments
by the antibodies was assessed through PCR reactions in
the linear stage of amplification using primers for RH
(5¢-ACATTTCCTCCCCTCCCCATCATAGTCCCT-3¢ and
5¢-ACACCCGCCAAAGGCCTTATCTCAG-3¢), GAPDH
primers or negative control primers
SiRNA interference
We prepared the dsRNA constructs pSilencerALY45-64
and pSilencerALY285-303 against the human Aly⁄ REF
gene by cloning inverted repeat sequences into pSilencer
2.0-U6 (Ambion Inc., Austin, TX, USA) The dsRNA
template consisted of 19 bp target sequences derived from
Aly⁄ REF mRNA and a 9 bp linker (TTCAAGAGA) for
transcription of the short hairpin dsRNA The construct
numerals of pSilencerALY45-65 and pSilencerALY285-303
corresponded to the nucleotide number of Aly⁄ REF
mRNA (GenBank accession no AF047002) A plasmid
with a random sequence was used as a negative control
dsRNA The pSilencer constructs were cotransfected into
HEL cells with pGL3-RH-158, pGL3-RH-238 or the
pGL3-SV40 promoter and the pRL-CMV vector Relative
luciferase assays were performed 24 h thereafter, which
was determined as the optimum by a time course
experi-ment
The HEL cells were also transfected solely with the
pSilencer constructs Twenty-four hours later, Aly⁄ REF
and Rh transcripts were quantified by real-time PCR using
SYBR Green PCR Master Mix and an ABI PRISM
7900HT (Applied Biosystems, Foster City, CA, USA) The
primer sequences to detect Aly⁄ REF and Rh transcripts
were for Aly⁄ REF: sense, 5¢-CTGGTCGCAGCTTAGG
AACAG-3¢ and antisense, 5¢-AATGTTCATGGGGCGGC
CATC-3¢, for RH: sense, 5¢-GCAACGATACCCAGTTT
GTC-3¢ and antisense, 5¢-AGTTGACACTTGGCCAGA
AC-3¢ The relative amounts of Aly ⁄ REF and RH were
assessed as differences in the threshold of the amplification
curve of the target gene from the internal control, GAPDH
(delta Ct), and in the delta Ct from the control siRNA
construct (delta-delta Ct)
Acknowledgements
We are grateful to Mr T Oyamada and Ms T Hatano
for valuable technical assistance This work was
supported by Grants-in-Aid for Scientific Research for the Ministry of Education, Science and Culture of Japan (Nos 15590587 for Dr H Okuda, 15591018 for
Dr T Kamesaki and 15590586 for Dr S Iwamoto)
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