Our results showed that: a the 1.7 kb fragment was a powerful activator of the reporter gene in human hepatoblastoma HepG2 and human embryonic rhabdomyosarcoma RD cell lines; b 512 bp up
Trang 1binding to the P1 promoter sequences of the human
AbH-J-J locus
Giordana Feriotto1, Alessia Finotti2, Giulia Breveglieri1, Susan Treves3, Francesco Zorzato4and Roberto Gambari2
1 Biotechnology Center, University of Ferrara, Italy
2 Department of Biochemistry and Molecular Biology, Section of Molecular Biology, University of Ferrara, Italy
3 Departments of Anaesthesia and Research, Kantonsspital, Basel, Switzerland
4 Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, Italy
Keywords
aspartyl (asparaginyl) b-hydroxylase;
humbug; junctate; specific transcription
factors; transcription
Correspondence
R Gambari, Department of Biochemistry
and Molecular Biology, University of Ferrara,
Via Borsari 46, 44100 Ferrara, Italy
Fax: +39 532 202723
Tel: +39 532 424443
E-mail: gam@unife.it
(Received 22 May 2007, revised 29 June
2007, accepted 3 July 2007)
doi:10.1111/j.1742-4658.2007.05976.x
Alternative splicing of the locus AbH-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) b-hydroxylase, humbug and junctate (truncated homologs of aspartyl (asparaginyl) b-hydroxylase with
a role in calcium regulation), and junctin (a structural protein of the sarco-plasmic reticulum membrane) Aspartyl (asparaginyl) b-hydroxylase and humbug are overexpressed in a broad range of malignant neoplasms We have previously reported the gene structure of this locus, showing the pres-ence of two putative promoters, P1 and P2, and characterized the P2 sequences, directing tissue-specific transcription of junctin, aspartyl (aspar-aginyl) b-hydroxylase and junctate In addition, aspartyl (aspar(aspar-aginyl) b-hydroxylase and humbug are expressed from exon 1 by the P1 promoter The present study identifies and functionally characterizes the P1 promoter activity of the AbH-J-J locus We demonstrate that mRNAs from the P1 promoter are actively transcribed in all the human tissues and cell lines analyzed, and define the transcription start point in HeLa and RD cells
To investigate the transcription mechanism we cloned 1.7 kb upstream of exon 1 from a human BAC clone, and produced progressively deleted reporter constructs Our results showed that: (a) the 1.7 kb fragment was a powerful activator of the reporter gene in human hepatoblastoma (HepG2) and human embryonic rhabdomyosarcoma (RD) cell lines; (b) 512 bp upstream of the transcription start site were essential for maximal promoter activity; and (c) progressive deletions from ) 512 resulted in gradually decreased reporter expression The region responsible for maximal tran-scription contains at least 12 GC boxes homologous to binding sequences
of specific transcription factor 1 (Sp1); by electrophoretic mobility shift assay and supershift analysis, we identified three GC-rich elements that bind Sp transcription factor family nuclear factors with very high effi-ciency A functional role of Sp transcription factors in upregulating P1-directed transcription was demonstrated by analysis of the effects of: (a) in vitro mutagenesis of the Sp1 transcription factor binding sites; (b) transfection with Sp transcription factor 1⁄ 3 expression vectors; and
Abbreviations
AAH, aspartyl (asparaginyl) b-hydroxylase; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; MEF-2, myocyte enhancer factor 2; Sp1, specific transcription factor 1; Sp3, specific transcription factor 3; TF, threshold fluorescence; TFD, transcription factor decoy.
Trang 2We have previously characterized the human AbH-J-J
locus, a genomic sequence that generates functionally
distinct proteins [1] In addition to the enzyme aspartyl
(asparaginyl) b-hydroxylase (AAH), this locus encodes
junctin, a structural protein of the sarcoplasmic
reticu-lum, and the truncated homologs of AAH, the Ca2+
-binding proteins humbug and junctate [1,2] AAH
catalyzes post-translational hydroxylation of aspartate
and asparagine residues in certain epidermal growth
factor-like domains present in a number of proteins,
including receptors and receptor ligands, involved in
cell growth and differentiation, as well as extracellular
matrix molecules [3] AAH, mediating cell motility and
invasiveness, is of interest because of its role in
placen-tal implantation and ‘receptivity’ of endometrium [4]
Humbug is a truncated homolog of AAH that lacks a
catalytic domain Overexpression of humbug increases
intracellular Ca2+levels by promoting its release from
intracellular stores [5] The levels of humbug
immuno-reactivity are directly associated with colon cancer
tumor grade and inversely associated with patient
sur-vival [5] AAH and⁄ or humbug are overexpressed in
infiltrative intrahepatic cholangiocarcinomas,
metasta-sized lung, breast, and colon, hepatocellular
carcino-mas, and malignant neuroectodermal tumors [6–10]
These proteins can contribute to the malignant
pheno-type by increasing motility and enhancing
prolifera-tion, survival, and cell cycle progression Inhibition of
AAH expression and its truncated homolog could
rep-resent an attractive approach for gene therapy of
infil-trating tumors [3,10]
Junctate is an integral Ca2+-binding protein of the
sarco(endo)plasmic reticulum membrane that forms a
supramolecular complex with the inositol
1,4,5-tris-phosphate receptor and modulates Ca2+entry through
receptor- and store-activated channels [1,11,12]
Our group previously reported the identification of
two putative promoter sequences, present within the
human AbH-J-J locus (named P1 and P2), that are
expected to regulate the transcription of this locus
[1,13] The generated primary transcripts are subjected
to alternative splicing and direct the synthesis of AAH,
humbug, junctin, and junctate [1,13] We have recently
reported the characterization of the P2 promoter, demonstrating that the myocyte enhancer factor 2 (MEF-2) transcription factor binds this promoter sequence and drives tissue-specific expression, being responsible for inducing transcription during muscle differentiation [1,13]
As little is known, to date, about the role of the P1 promoter, in this work we focused our attention on this regulating sequence To characterize the expression directed by the P1 promoter, we analyzed the corre-sponding mRNAs in different human tissues and cell lines Furthermore, transfections of human hepatoblas-toma (HepG2) and human embryonic rhabdomyosar-coma (RD) cells with progressively deleted reporter constructs of the 1.7 kb DNA sequence upstream of exon 1 were performed, and P1 promoter sequences were characterized by analyzing the transcriptional activity of each fragment As several homologies to specific transcription factor 1 (Sp1)-binding sites were found, by computer-assisted analysis, within the pro-moter sequence exhibiting maximal transcriptional activity, the interactions of these GC-rich elements with factors belonging to the Sp1 family were studied
by electrophoretic mobility shift assay (EMSA) [14,15] Molecular interaction studies were also performed
in vitro by supershift assays, and in intact cells by chromatin immunoprecipitation (ChIP) Functional assays were performed by in vitro mutagenesis of the Sp1-binding sites, by cotransfection with Sp1⁄ Sp3 expression vectors, and by cell treatment with decoy oligonucleotides targeting Sp transcription factors
Results
Transcriptional organization of the human AbH-J-J locus and identification of the transcription
initiation sites proximal to the P1 promoter The structural organization of the human AbH-J-J locus
is shown in Fig 1 The scheme presented is based both
on results previously reported in detail elsewhere [1,13] and on newly performed studies employing RT-PCR The combination of data obtained by PCR
(c) treatment with decoy oligonucleotides targeting Sp transcription factors
In addition, Sp1 and Sp3 transcription factor chromatin immunoprecipita-tion demonstrated in vivo binding of these proteins to P1 promoter Our results suggest that Sp transcription factors positively regulate the core of the P1 promoter, and the comparison of the two promoters of the AbH-J-J locus demonstrates that they are very different with regard to transcrip-tional efficiency and ability to direct tissue-specific transcription
Trang 3amplification and sequencing allowed us to define the
splicing events (Fig 1) as well as the structure of the
5¢-region of this locus [1] The data obtained indicate
that the use of different splice donors is involved in the
generation of protein diversity by alternative splicing
(see lower part of Fig 1) [1,2] Furthermore, the P1
promoter sequences direct the expression of these
pro-teins in most human tissues [2,13] The RT-PCR
approach presented in Fig 2A shows that by using an
exon 1-specific forward primer and an exon 3-specific
reverse primer, we were able to amplify all the tran-scripts starting from the P1 promoter (AAH, humbug)
We employed this RT-PCR approach to examine sam-ples of total RNA from several human adult tissues, and confirmed by DNA sequencing the fidelity of the PCR products The results obtained from pancreas, brain, adrenal gland, liver, heart and skeletal muscle show that transcription directed from the P1 promoter occurs in all the tissues analyzed (Fig 2B) In contrast, we have previously shown that the expression directed by the P2 promoter is tissue specific, as a high level of transcrip-tion is present, in particular, in skeletal muscle, cardiac muscle, and brain [2,13] The P1 promoter directs tran-scription also in RD, HepG2, human breast cancer (MCF7), human cervix epithelial carcinoma (HeLa) and human embryonic kidney (Hek293) tumor cell lines (Fig 2B)
As the transcription start site (TSS) from the P1 pro-moter has not been previously described, we also per-formed 5¢-RACE in order to precisely map the origin
of transcription Our experiments were performed on 5¢-capped mRNA isolated from HeLa and RD cells As shown in Fig 3A, lanes a and b, a prevalent band is evident following electrophoresis of 5¢-RACE products
We cloned the gel-purified PCR products, and their characterized nucleotide sequences (Fig 3B, arrows) allowed mapping of multiple TSSs with differential strength The major TSS, resulting from the stronger band in the PCR gel, was designated + 1 (Fig 3B, larger arrow) The nucleotide sequences located upstream from these TSSs were considered as potential regulatory regions belonging to the P1 promoter
Fig 1 Structure of the 5¢-end of the human locus for AAH, junctin, junctate, and humbug Arabic numbers over black boxes indicate exons Intervening sequences are indicated by Roman numerals The two putative promoters P1 and P2 are indicated A schematic representation
of AAH, junctin, junctate and humbug exon splicing is given at the bottom of the panel The cytoplasmic, transmembrane, positively charged,
Ca2+-binding and catalytic domains are indicated The locations of AUG, stop codons, and poly(A) signals are shown The PstI (P) plasmid subclone of BAC 1 [1] covering the first exon of the locus is also shown.
A
B
Fig 2 RT-PCR analysis of the transcripts starting from the P1
pro-moter in adult human tissues or cell lines (A) The exon 1 starting
mRNAs of AAH and humbug, the PCR primers and the 134 bp
PCR product are represented; gray boxes indicate exons common
to the two transcripts (B) Electrophoresis analysis of oligo-dT
RT-PCR products obtained with the e1F ⁄ e3R primers in the absence
of template (a) or in the presence of cDNA from human adult
nor-mal tissue (b, pancreas; c, brain; d, adrenal gland; e, liver; f, heart;
g, skeletal muscle) or cell line (h, RD; i, HepG2; j, MCF7; k, HeLa;
l, Hek293) total RNA M, pUC Mix Marker 8 (Fermentas).
Trang 4Transcriptional activity of the AbH-J-J P1
promoter
To test whether the exon 1 5¢-flanking sequences have
promoter activity, we cloned a 3.1 kb partially digested
PstI fragment (Fig 1) from the human chromosome 8 BAC 1 clone [1] The PCR-generated fragment span-ning from) 1683 to + 81 with respect to the principal TSS was then inserted into the firefly luciferase repor-ter vector pGL3-basic, sequenced, and used to generate progressively deleted constructs for transient transfec-tion experiments [13] The reporter constructs (A–M), generated as described in Experimental procedures, are shown on the left side of Fig 4A We then tested the ability of these constructs to drive transcription of luciferase in HepG2 cells and RD cells Promoter activity was expressed as fold induction relative to that
of cells transfected with pGL3-basic vector (right side
of Fig 4A,B) Our results show that the largest frag-ment () 1683 ⁄ + 81) exhibited high reporter gene expression in the HepG2 and RD cell lines (the fold induction is about 100 and 45, respectively) Removal
of the ) 1683 ⁄ ) 834 sequence significantly increased promoter activity (compare A and E constructs of Fig 4A,B) Further removal of the ) 834 ⁄ ) 512 frag-ment preserved transcriptional activity to comparable levels in the analyzed cell lines (E–H constructs, Fig 4A,B); however, deletion to nucleotide ) 389 resulted in a significant decrease of luciferase activity (I construct) and, when the sequence was progres-sively removed to ) 240 and ) 160, reporter expression
A B C D E F G H I L M
A
Relative luciferase activity 0
+81 -1683
-1289 -1204 -1017 -834 -661 -634 -512 -389 -240 -160
Relative luciferase activity
Relative luciferase activity
0 6,212,5
-512/+81 P1 sequences (H) -265/+115 P2 sequences
HepG2
100
B C D E F G H I L M
A
B
C
Fig 4 AbH-J-J P1 promoter activity in HepG2 and RD cell lines (A) HepG2 cells were transiently transfected with sequentially deleted reporter constructs of the ) 1683 ⁄ + 81 P1 nucleotide sequence (represented on the left side of the figure, A–M) Transient transfection and luciferase assays were performed in triplicate; the data (right side of the figure) were normalized to Renilla luciferase activity, and are shown
as relative activities compared to that for pGL3-basic, a reporter vector with a basal promoter The values are the means ± SD of at least three independent experiments (B) The same procedure as in (A) was performed with the RD cell line (C) AbH-J-J P1 and P2 promoter activity in the RD cell line Cells were transiently transfected with the reporter constructs that present the maximal transcriptional activity of each promoter [13].
a P1 transcription initiation sites
183 bp
Fig 3 P1 transcription initiation site mapping (A) 5¢-RACE analysis
of AbH-J-J locus exon 1 starting transcripts cDNAs, synthesized
from HeLa and RD cell total RNA, were amplified with the
gene-specific e3R primer The nested PCRs were performed with the
gene-specific e1R primer, complementary to exon 1, and one-fifth
of the reactions, derived from HeLa (a) and RD (b) cells, were
ana-lyzed by gel electrophoresis M, pUC Mix Marker 8 (B) Nucleotide
sequence of 5¢-RACE products The most represented nested PCR
products were gel-purified, cloned and sequenced The principal
TSSs are shown by arrows (the stronger TSS was numbered + 1).
The gene-specific reverse primer used for the last PCR is
under-lined, and the translation start site (ATG) is indicated.
Trang 5gradually decreased (L and M constructs) In
conclu-sion, our findings indicate that 512 bp upstream of the
TSS are essential for maximal promoter activity, and
deletions to) 389, ) 240 and ) 160 resulted in
progres-sive reduction of transcription to about 75%, 40%,
and 7%, respectively
In addition, when we compared the maximal
activi-ties of P1 and P2 promoter sequences in inducing
tran-scription of RD cells, the fold inductions were
dramatically different, the P1 promoter being 16-fold
more active (Fig 4C) It should be underlined that the
P1 promoter sequences do not share homology regions
and signals for transcription factors with the P2
pro-moter For instance, no MEF-2-binding sites are
pres-ent within the P1 promoter; this finding is of interest,
when related to the different tissue specificities of these
two promoters [1,13,16]
Identification of GC boxes within the) 512 ⁄ + 33
AbH-J-J P1 promoter region
Sequence analysis of the region required for maximal
expression from the P1 promoter was performed using
the tfsearch program Looking for homology to
known signals for transcription factors and imposing
an 80% threshold, we identified within the ) 512 ⁄ + 33 region at least 12 GC-rich boxes similar to the Sp1 consensus binding sequence (Fig 5A) The location of these putative Sp1-binding sites within the P1 pro-moter sequence is shown in Fig 5A,B, together with the transcription initiation sites and the ATG signal
As the region responsible for maximal P1 promoter transcription contains GC-rich elements (H construct, Fig 4), we concentrated our attention on the activity
of binding of nuclear extract to these boxes
Binding of nuclear factors to GC-rich boxes
of the AbH-J-J P1 promoter
In order to study protein–DNA interactions and fur-ther characterize the transcription factors involved, we performed competitive EMSA [13] Table 1 and Fig 5A show the synthetic oligonucleotides used for the bandshift experiments The results obtained using
2 lg of HepG2 cell nuclear extract and the32P-labeled Sp1mer double-stranded oligonucleotide, which con-tains the consensus-binding site for Sp1 transcrip-tion factor, are shown in Fig 6A [17,18] The probe
A
B
Fig 5 DNA sequence of the AbH-J-J P1 promoter region (A) ) 512 ⁄ + 112 P1 pro-moter and 5¢-UTR sequences Solid and dashed lines indicate the oligonucleotides used in EMSA (Table 1) The sequences homologous to Sp1 transcription factor-bind-ing site are boxed; the percentage homol-ogy was obtained with TF SEARCH version 1.3 Arrows indicate the characterized tran-scription initiation sites The 5¢-end nucleo-tide positions of the progressively deleted promoter sequence present in the reporter constructs are shown in gray (B) Schematic representation of the ) 512 ⁄ + 112 region of the P1 promoter Elements homologous
to the Sp1-binding site are indicated by boxes, the nucleotide deletions of reporter constructs are shown in gray.
Trang 6interacts with nuclear proteins producing the three
retarded complex pattern typical of transcription
fac-tors belonging to the Sp family A high-mobility band
and two overlapping low-migrating bands (Fig 6A,
lane 1) are generated As expected, a 100-fold excess
of unlabeled Sp1mer oligonucleotide completely
abol-ished sequence-specific interactions of the nuclear
pro-teins with the probe (Fig 6A, lane 2) Competitive
experiments performed using unlabeled
oligonucleo-tides containing the previously identified GC-rich
boxes of the P1 promoter region (Table 1)
demon-strated that F⁄ Gmer, H ⁄ Imer and D ⁄ Emer interfere
with the formation of the three complexes (Fig 6A,
lanes 3, 4 and 7) These results suggest that the last
three oligonucleotides contain binding elements
recog-nized by Sp family transcription factors [19] On the
other hand, competitive bandshift demonstrated that
the three Sp bands were only slightly reduced by
Kmer and Lmer (Fig 6A, lanes 5 and 6), whereas
Jmer did not decrease the abundance of the
Sp-spe-cific complexes (Fig 6A, lane 8) To better
character-ize the binding efficiency of the oligonucleotides under
investigation, we performed bandshift with the same
probe and different fold molar excesses of competitors
(Fig 6B,C) As observed for Sp1mer (Fig 6B, lane
10), the three complexes were completely disrupted by
a six-fold molar excess of unlabeled H⁄ Imer (Fig 6B,
lane 15), whereas the same excess of F⁄ Gmer
decreased the binding to about 5% of the control in the absence of competitor (Fig 6B, lane 12) Further-more, the competition with 50-fold molar excess of unlabeled D⁄ Emer, Kmer and Lmer decreased the interactions to 12%, 60% and 75% of the control, respectively (Fig 6C, lanes 20, 22 and 24), whereas Jmer competitor was not active even if used at 100-fold molar excess (Fig 6C, lane 26) To further con-firm whether the previously identified GC-rich P1 sequences are able to bind Sp family transcription fac-tors, we performed bandshift assays using as probe the oligonucleotides under investigation, which gener-ated the same complex migration profile obtained with labeled Sp1mer Figure 6D shows an example of the interactions of nuclear extracts with H⁄ Imer probe As expected from our previous assays, the three complexes were completely disrupted by an excess of the competitors Sp1mer, F⁄ Gmer and
H⁄ Imer (Fig 6D, lanes 28–33), but not by Jmer or the unrelated oligonucleotide MyDmer (Fig 6D, lanes
34 and 35) [13]
Supershift with Sp1 and Sp3 transcription factor antisera
In order to obtain the formal demonstration of an involvement of Sp-related proteins in molecular inter-actions at the P1 promoter, supershift experiments
Table 1 Double-stranded synthetic oligonucleotides.
Oligonucleotides used for EMSA
Q-PCR primers
P1 ChIP F
P1 ChIP R
ACGAACCTGTGACTCCCTCCCG Neg ChIP F
Neg ChIP R
CCAGCCTCTTCCATTGGATACAA e1 F
e5 R
AATAAAACTTTGGCATCATCCACTCAAAATCTCC HMBS F
HMBS R
GGTAGCCTGCATGGTCTCTTGTAT
a
Nucleotide mutations are underlined. bThe oligonucleotide containing the Sp1 site was purchased from Geneka (Montreal, Canada).
c Feriotto et al [13] d HMBS, hydroxymethylbilane synthase.
Trang 7were performed using the previously identified GC-rich
boxes As shown in the representative experiment
reported in Fig 7A, antibodies against Sp1 (lane 2)
and Sp3 (lane 3) transcription factors supershifted the
specific complexes, indicating that these proteins are
able to bind in vitro to P1 promoter sequences of the
AbH-J-J gene locus
ChIP assay
In order to verify whether the binding of Sp1 and Sp3
proteins to the AbH-J-J P1 promoter occurs in intact
cells, ChIP was performed HeLa cells were fixed,
chromatin was immunoprecipitated with antibodies
against Sp1 or Sp3 transcription factors, and
quantita-tive real-time PCR was performed on recovered DNA
using primers amplifying a P1 promoter region of
the AbH-J-J gene locus that contains the previously
characterized Sp1-d⁄ e, Sp1-f ⁄ g and Sp1-h ⁄ i boxes
(Fig 5B) As an immunoprecipitation control,
nonim-mune rabbit serum or unrelated antisera, of the same
isotype, recognizing MEF-2A and myogenin were
used Figure 7B shows a representative example of amplification curves (in duplicate determinations) obtained by SYBR Green real-time PCR The data demonstrate that HeLa chromatin immunoprecipitated using Sp1 or Sp3 antiserum reaches the ‘threshold fluo-rescence’ (TF) value about six cycles before nonim-mune serum ChIP samples The results obtained with ChIP performed with antibodies against MEF-2A (Fig 7B) and myogenin (data not shown) are similar
to those of immunoprecipitation controls obtained using nonimmune rabbit serum PCR was also per-formed using control primers flanking a genomic region about 5 kb upstream of the P1 promoter (Table 1) Because this sequence, lacking Sp1-binding sites, should not be bound by Sp factors, PCR with the negative control primers was used to normalize quantitative results from different immunoprecipita-tions
For data analysis, we followed the methodology described in Experimental procedures Normalized results, reported in Fig 7C, indicate mean increases in amplification signal of 13-fold (Sp1 ChIP) and 22-fold
fold
molar
excess of competitor
fold
molar
excess of competitor
fold molar excess of competitor
fold molar excess of competitor
Sp3
Sp3 Sp1/3
Sp1/3
–
H/Imer
Probe
–
27 28 Sp1
mer
F/G
mer
H/I
mer
H/I
mer
D/E
mer
mer
–
Sp1mer
Probe
Sp1
mer
Sp1
mer
F/G
mer
H/I
mer
F/G
mer
H/I
mer
29 30 31 32 33 34 35
6
*
Sp3
Sp1/3
*
12 6 12 25 6 12 25
9 10 11 12 13 14 15 16 17
18 19 20 21 22 23
100
24 25 26
25 50 25 50 100 100 50
100 100 50 100 100
Fig 6 AbH-J-J promoter P1 elements homologous to the Sp1 box bind HepG2 nuclear factors EMSAs were carried out on
2 lg of HepG2 nuclear extracts as described
in Experimental procedures, using Sp1mer (A–C) or H ⁄ Imer (D) probe (Table 1) –, probe was incubated with nuclear extracts
in the absence of competing oligonucleo-tides The fold molar excess of the added competitor (Table 1) is reported at the bot-tom of each panel Arrows and asterisks indicate the specific and nonspecific com-plexes, respectively.
Trang 8(Sp3 ChIP) when PCR was performed with P1
promoter-specific primers relative to PCR products
obtained with negative control primers These data
strongly indicate that transcription factors belonging
to the Sp1 superfamily interact with the P1 promoter
in intact HeLa cells
Effects of mutations of Sp1-binding sites
on P1-directed transcription
In order to verify the role of Sp1-binding sites in
P1-directed transcription, we first designed D⁄ Emut and
F⁄ Gmut mutant oligonucleotides (Table 1) that are
unable to bind the Sp family factors We demonstrated
that mutated D⁄ Emut and F ⁄ Gmut failed to disrupt
D⁄ Emer–Sp protein complexes (Fig 8A, lanes 1 and
5) In agreement with this, no retarded bands were
generated following incubation of nuclear factors with
D⁄ Emut and F ⁄ Gmut probes (Fig 8B, lanes 7 and 8) Accordingly, we produced, starting from the wild-type reporter construct (H, ) 512 ⁄ + 81), two plasmids car-rying the previously characterized Sp1-binding site mutations (d⁄ e mut and f ⁄ g mut) When the mutant constructs were used to transfect HeLa cells, 22% and 12% decreases in luciferase activity (relative to the wild-type H construct) were detected (Fig 8C) As expected, when transfection was performed with the double mutant construct (d⁄ e + f ⁄ g mut), a further decrease of transcription activity was consistently found () 32%) These results demonstrate that muta-tions of Sp1-binding sites lead to a decrease of tran-scription activity, suggesting a functional role of Sp1
in promoting P1-directed transcription of the AbH-J-J gene locus
0
Sp1/3
1
–
D/Emer
Probe
Ab-Sp3 Ab-Sp1
Sp3
Negative control
Fold increase over negative control PCR
Cycle number
16 18 20 22 24 26 28 30 32 34 36
Nonimmune
MEF2A ChIP
Input
TF
10 1
10 2
10 3 P1 promoter Q PCR
Sp1 ChIP Sp3 ChIP
38
Negative control
P1 promoter Sp1 ChIP
*
*
5
A
C
B
Fig 7 Interaction of Sp1 and Sp3 transcription factors with the AbH-J-J P1 promoter (A) A supershift assay was performed using D ⁄ Emer probe and 2 lg of nuclear extract from HeLa cells; the probe was incubated with nuclear exctract in the presence of antibodies (Ab) against Sp1 or Sp3 factors –, control sample in the absence of antibody Arrows and stars indicate the specific and supershifted complexes, respec-tively (B) Quantitative real-time PCR profiles for the amplification of the P1 promoter are shown for a representative ChIP assay in which chromatin from HeLa cells was immunoprecipitated using either Sp1 and Sp3 antiserum The data (from duplicate determinations) demon-strate the early exponential increase in fluorescence as a result of SYBR Green I incorporation into the amplifying P1 promoter fragment Sp1 ChIP, Sp3 ChIP and 2A ChIP indicate duplicate curves from chromatin that have been immunoprecipitated with Sp1, Sp3 or MEF-2A antiserum, respectively; nonimmune serum indicates curves from immunoprecipitations with nonimmune rabbit serum Input represents curves obtained from HeLa chromatin (1%) before immunoprecipitation The cycle at which the amplification curve reaches threshold fluo-rescence (TF), the threshold cycle, were used to determine the relative amounts of promoter in each sample (C) In vivo association of Sp1 and Sp3 transcription factors with the AbH-J-J P1 promoter The results, obtained from ChIP assay quantitative real-time PCR using Sp1 (Sp1 ChIP) or Sp3 (Sp3 ChIP) antiserum, were analyzed following the methodology described in Experimental procedures The fold increase over negative control PCR in each case compares the values obtained by P1 promoter amplification with the corresponding amplification of
a distal genomic region lacking Sp1-binding sites All data represent the mean of two determinations in triplicate from each of at least two independent immunoprecipitations The asterisk indicates that the value is significantly different (P < 0.05) from the control value.
Trang 9Transcriptional effect of Sp1 and Sp3 expression
vectors on P1 promoter activity
More functional evidence for the involvement of
Sp1-related proteins in P1-directed transcription was
obtained after cotransfection of Drosophila
melano-gaster SL-2 cells with the H () 512 ⁄ + 81) reporter
construct and pPAC-Sp1 and pPAC-Sp3 expression
vectors As control, SL-2 cells were cotransfected with
pPAC empty vector The SL-2 cells were chosen
because they do not contain Sp proteins [20] and are,
for this reason, employed in functional studies focused
on these transcription factors The results obtained
indicate a significant increase of P1-directed
transcrip-tion in SL-2 cells transfected with pPAC-Sp1 and
pPAC-Sp3 (Fig 9, + 45%) These data suggest that
Sp1 and Sp3 proteins play a positive role in
P1-direc-ted transcription In agreement with this, triple
trans-fection of SL-2 cells with the H reporter construct, and
the pPAC-Sp1 and pPAC-Sp3 expression vectors,
pro-duced higher levels of P1-directed transcription (Fig 9,
+ 130%)
Effect of a decoy oligonucleotide targeting Sp family transcription factors on P1 promoter-specific mRNA levels
In order to further confirm the role of Sp1-related pro-teins in the transcription regulation of the AbH-J-J locus, a transcription factor decoy (TFD) approach was employed This approach is based on the use of double-stranded oligonucleotides for targeting tran-scription factors, with consequent inhibition of their interactions with promoters The TFD approach has been demonstrated to be very useful in inhibiting gene expression when targeted at key transcription factors [21] We employed as a TFD molecule the double-stranded oligonucleotide Sp1mer reported in Table 1 Briefly, we transfected HeLa cells with 2 lgÆmL)1 of Sp1mer double-stranded oligonucleotide, or a scram-bled sequence of the same length as a control Twenty-four hours later, RNA extraction was performed and the transcripts relative to P1 promoter activity were analyzed by using quantitative real-time RT-PCR The results obtained demonstrate that in cells treated with the Sp1 decoy, a 64% reduction of transcription direc-ted by the P1 promoter was obtained (Fig 10) In con-trast, scrambled oligonucleotide exibited no effects on transcription of the analyzed mRNA (Fig 10)
Discussion
The aim of the present work was to investigate in detail one of the two putative promoter sequences regulating the transcription of the AbH-J-J locus [1,13] We iso-lated and identified the 5¢-flanking region of exon 1 of this locus The cloned nucleotide sequence allowed us to characterize the P1 promoter region, which is involved
in the regulation of AAH and humbug expression
We have been able to identify transcripts relative to P1 promoter activity in all tissues and cell lines ana-lyzed [2,13,16] Similar to many housekeeping gene
Relative Luc Activity
Sp1/3
6
Probe
7 8
1 2
–
D/Emer
D/E
met
D/E
mer
D/E
met
D/E
mut
mut
F/G
mut
F/G
mer
3 4 5
Sp3 Sp1/3
Sp3
0
H (-512/+81)
d/e mut
f/g mut
d/e + f/g mut
A
C
B
Fig 8 Mutational analysis of Sp1 elements in the AbH-J-J P1
pro-moter (A–B) EMSAs were performed using D ⁄ Emer probe or
mutant probes (D ⁄ Emut, F ⁄ Gmut) and 2 lg of nuclear extract from
HepG2 cells Probes were incubated with nuclear extracts in the
absence or in the presence (A) of 100-fold molar excess of
compet-ing oligonucleotides (Table 1) Arrows indicate the specific
complexes (C) HepG2 cells were transfected with wild-type H
( ) 512 ⁄ + 81), single mutant Sp1-d ⁄ e element (d ⁄ e mut) and
Sp1-f ⁄ g element (f ⁄ g mut) or double mutant (d ⁄ e + f ⁄ g mut)
AbH-J-J P1 promoter reporter constructs Transient transfection
and luciferase assay were performed in triplicate, and the data
were normalized to Renilla luciferase activity and reported as ratios
(means ± SD) to the wild-type reporter construct H.
Relative Luc Activity (fold increase) 0
pPac pPac Sp1 pPac Sp3 pPac Sp1/Sp3
0.5 1 1.5 2 2.5
+ 45%
+ 130% + 45%
Fig 9 Cotransfection of Drosophila SL2 cells with the P1 promoter reporter construct and the Sp1 and ⁄ or Sp3 expression vectors Dro-sophila SL2 cells were cotransfected with ) 512 ⁄ + 81 P1 promoter reporter construct (H) in the presence of Sp1 (pPac Sp1) and ⁄ or Sp3 (pPac Sp3) expression vectors The pPAC void vector was used as cotransfection control plasmid Results (mean ± SD) are presented as fold increase in luciferase activity for cotransfection over that for the promoter construct alone (pPAC) from three experiments, each performed in triplicate.
Trang 10promoters, the region under investigation lacks a TATA
box and an initiator element [22,23] In contrast, this
sequence is GC-rich and presents homologies with the
Sp1 consensus binding site, in agreement with previous
studies showing that the transcription of other
TATA-less promoters frequently involves the action of
proxi-mal Sp1 sites [24,25] The mapping of the initiation of
transcription using 5¢-RACE revealed the presence of
different TSSs located around position) 110 relative to
the translation initiation start in HeLa and RD cells
We found, upstream of exon 1, cis-elements with
nega-tive and posinega-tive effects on transcription Furthermore,
the maximal promoter is located within 512
nucleo-tides of the principal TSS Computer analysis indicates
the presence of at least 12 sites that match the
struc-tural determinants of Sp1-binding specificity, and the
screening by EMSA demonstrated three GC-rich
ele-ments that bind, with high efficiency, transcription
fac-tors belonging to the Sp family [14,15] The migration
profiles of the complexes produced by nuclear extracts
binding to our GC-rich elements resemble the
well-known electrophoresis pattern obtained with the
con-sensus binding site for Sp1 [17–19] Sp1 is a ubiquitous
DNA-binding protein that activates the transcription
of many cellular and viral genes [26,27] Other
tran-scription factors, Sp2–Sp6, have been described that
have similar structural properties and DNA-binding
specificities as Sp1 [14,15] Sp1 and Sp3 are the major
DNA-binding constituents observed in nuclear extracts
with Sp1 consensus element in EMSA [28] The actions
of Sp1 and Sp3 at a given promoter appear to be
com-plex, but, in many cases, expression of Sp3 is thought
to antagonize the stimulatory actions of Sp1 on gene
transcription [29,30] Moreover, Sp3 can act as an
acti-vator or repressor of Sp1-mediated activation,
depend-ing on the sequence context and the availability of
specific coactivators, corepressors or other transcrip-tion factors [27,28] The involvement of Sp1⁄ Sp3 bind-ing was confirmed by supershift experiments and ChIP assays The effect of the Sp proteins on transcription can be influenced by multiple factors, including phos-phorylation, redox state, and acetylation [31] The first conclusion of the present article is that the P1 pro-moter of the AbH-J-J locus contains Sp1 cis-acting motifs that are putatively involved in the extensive transcription directed by this promoter
The second conclusion is that these interactions are functionally relevant for transcriptional control The relevance of Sp1- and Sp3-binding sites for the tran-scription regulation of the P1 promoter of the AbH-J-J locus has been addressed with three complementary approaches: (a) mutagenesis of Sp1 elements; (b) trans-fection of Sp-null SL-2 cells with Sp1⁄ 3 expression vec-tors; and (c) use of a TFD approach targeting Sp factors
The results obtained also demonstrate that Sp1 tran-scription factors and Sp1-binding sites are involved in the upregulation of the P1 promoter of the human AbH-J-J gene locus Although our results do not con-clusively show the involvement of other factors in the transcription of the AbH-J-J locus, we demonstrate that some of the Sp1-binding activities are important for transcription directed by the P1 promoter
When the P1 and P2 promoter sequences of the AbH-J-J locus are compared, important differences are clearly detectable [13,16] The most interesting result emerging from studies focused on the P2 promoter is that the Ca2+-dependent transcriptional factor MEF-2 activates the transcription of junctin, junctate and AAH in muscular tissues and brain [13] No Sp1-bind-ing sites are present in the P2 promoter In contrast, the P1 promoter directs the expression of AAH and humbug in many tissues and contains several function-ally active Sp1-binding sites [1,2,13] The finding that the sequences present in the upstream P1 promoter are significantly different from those of the P2 promoter
is, in our opinion, of great interest
In addition, our data do not exclude a concerted reg-ulation of the two promoter sequences based on inter-actions between different transcription factors There is strong evidence demonstrating that transcription fac-tors belonging to the Sp1 family interact with other transcription factors, including some proteins binding
to the P2 promoter [13,32,33] For instance, physical interactions between Sp1 and MEF-2 have been dem-onstrated in DNA-binding complexes formed in vitro
by nuclear extracts [34] An intriguing possibility to be further analyzed is the generation of a looping structure directed by physical interactions between the P1 and P2
Relative mRNA content 0
- ODN
*
Scramble ODN
Sp1 ODN
0.25 0.5 0.75 0.1 1.25 1.5
Fig 10 Effect of decoy oligonucleotide targeting of Sp transcription
factors on P1 promoter-specific mRNA levels HepG2 cells were
transiently transfected with Sp1mer double-stranded decoy
oligonu-cleotide (Sp1 ODN, Table 1) for 24 h or remained untreated
(– ODN) The effect of scrambled, unrelated oligonucleotide
(Scram-ble ODN) is also reported The cDNA obtained from total RNA was
subjected to quantitative real-time PCR for P1 promoter-specific
transcripts Results are representative of three independent
experi-ments carried out in triplicate; the DDC t method was used to
com-pare gene expression data, and standard error of the mean was
calculated Statistical significance: *P < 0.05.