activating protein 2a AP-2a regulate expression of human KCTD10 gene by binding to proximal region of promoter Rushi Liu1,*, Aidong Zhou1,*, Daolong Ren1,*, Ailan He1, Xiang Hu1, Wenfeng
Trang 1activating protein 2a (AP-2a) regulate expression of human KCTD10 gene by binding to proximal region of promoter Rushi Liu1,*, Aidong Zhou1,*, Daolong Ren1,*, Ailan He1, Xiang Hu1, Wenfeng Zhang1,
Liping Yang1, Mingjun Liu1, Hong Li1, Jianlin Zhou1, Shuanglin Xiang1and Jian Zhang1,2
1 Key Laboratory of Protein Biochemistry and Development Biology of State Education Ministry of China, Hunan Normal University, China
2 Model Organisms Division, Shanghai Second Medical University, China
Rat potassium channel tetramerization
domain-con-taining 10 (KCTD10) gene was recently cloned and
identified as a new member of the polymerase
delta-interacting protein 1 (PDIP1) gene family The amino
acid sequence of rat KCTD10 shares high levels of
identity with other members in this family; for
exam-ple, 65.1% identity with PDIP1 and 66.7% identity
with tumor necrosis factor alpha-induced protein 1
(TNFAIP1), respectively Like PDIP1 and TNFAIP1, KCTD10 protein also contains a BTB⁄ POZ domain and a potassium channel tetramerization (K-tetra) domain (a relative of BTB⁄ POZ domain) at its N-terminus, and a proliferating cell nuclear antigen (PCNA)-binding motif at its C-terminus [1]
PCNA, a multifunctional protein, plays critical roles in a variety of eukaryotic cellular processes,
Keywords
AP-2a; KCTD10; promoter; regulatory
element; SP1
Correspondence
J Zhang, Key Laboratory of Protein
Biochemistry and Development Biology of
State Education Ministry of China, College
of Life Sciences, Hunan Normal University,
Changsha, Hunan 410081, China
Fax: +86 731 887 2792
Tel: +86 731 887 2792
E-mail: Zhangjian@hunnu.edu.cn
*These authors contributed equally to this
work
(Received 25 October 2008, revised 7
December 2008, accepted 11 December
2008)
doi:10.1111/j.1742-4658.2008.06855.x
Potassium channel tetramerization domain-containing 10 gene (KCTD10) belongs to the polymerase delta-interacting protein 1 (PDIP1) gene family Mouse KCTD10 was thought to interact with proliferating cell nuclear antigen and the small subunit of polymerase d, and to have roles in DNA repair, DNA replication and cell-cycle control To better understand the regulatory mechanism of KCTD10 expression, we characterized the pro-moter of human KCTD10 containing a 639 bp fragment of the 5¢-flanking region ()609 ⁄ +30) A primer extension assay identified the transcription start site as an A, 63 bp upstream of the start codon The promoter region contains neither a TATA box nor a CCAAT box, but a CpG island was found near to the transcription start site Deletion mutagenesis showed that the region from )108 to +30 was indispensable to the promoter activity, and site-directed mutation analysis in this proximal promoter region of KCTD10revealed two important transcription regulatory elements of speci-ficity protein 1 (SP1) and activating protein-2 (AP-2) An in vivo chromatin immunoprecipitation assay further demonstrated that SP1 and AP-2a could bind to this proximal promoter region Moreover, using a luciferase repor-ter assay, quantitative real-time RT-PCR and wesrepor-tern blot analysis, both overexpression and RNA interference of SP1 and AP-2a indicated that the binding of SP1 to the proximal promoter region stimulated the promoter activity and endogenous KCTD10 expression, whereas binding of AP-2a to this region showed opposite effects
Abbreviations
AP-2, activating protein-2; ChIP, chromatin immunoprecipitation; DPE, downstream promoter element; KCTD10, potassium channel tetramerization domain-containing 10; PCNA, proliferating cell nuclear antigen; PDIP1, polymerase delta-interacting protein 1; Sp1, specificity protein 1; TNFAIP1, tumor necrosis factor alpha induced protein 1; TNF-a, tumor necrosis factor-alpha; TSS, transcription start site.
Trang 2including DNA replication, DNA repair and
cell-cycle progression by interacting with proteins
involved in these processes The best-understood role
of PCNA was as a slide clamp to tether DNA
poly-merase d to its template for high processive DNA
synthesis [2,3] Many proteins have been shown to
bind PCNA; they all contain a consensus
PCNA-binding motif, which was initially identified in p21
[4] Recent studies have shown that PDIP1 and
TNFAIP1 could interact with PCNA and stimulate
PCNA-dependent polymerase d activity [5,6]
KCTD10 can also interact with the small subunit of
DNA polymerase d (p50) and PCNA [1] These
find-ings suggested that PCNA might function as a
regu-latory target of the PDIP1 gene family including
KCTD10, coordinating DNA replication, DNA repair
and cell-cycle progression
Tumor necrosis factor-alpha (TNF-a) is a
multifunc-tional cytokine involved in a variety of biological
activities, such as apoptosis, proliferation, B-cell
acti-vation and some inflammatory responses [7] It is
known that TNF-a induces cell proliferation in liver
regeneration after hepatocyte loss caused by surgical
resection or chemical injury [8] Like other members of
the PDIP1 gene family, KCTD10 is also supposed to
be induced by TNF-a [1] KCTD10 is an interacting
partner of PCNA and the small subunit of pol d, so it
probably plays some roles in cell proliferation by
link-ing TNF-a signallink-ing to DNA synthesis Recently, a
transgenic Caenorhabditis elegans model of Alzheimer’s
disease was achieved by expressing human b-amyloid
peptide [9] In the brain of transgenic C elegans,
TNFAIP1 expression was found to be markedly
increased; and the brain region with minimum
patho-logical symptoms showed the highest expression of
TNFAIP1, suggesting that TNFAIP1 may have a
protective function during Alzheimer’s disease
progre-ssion [9] These studies indicated that the PDIP1 gene
family, including KCTD10, might function in the
pro-liferation and development of cells, or in disease
progression
Although the function of KCTD10 is very
impor-tant, regulation of its expression remains unclear We
characterized a 639 bp genomic fragment in the
5¢-flanking region of human KCTD10; and identified
the binding sites of two transcription factors,
specific-ity protein 1 (SP1) and activating protein-2a (AP-2a),
in the promoter region of KCTD10 We found that the
proximal promoter region from )108 to +30 was
indispensable for basal promoter activity of KCTD10;
and SP1 and AP-2a can regulate the promoter activity
and endogenous KCTD10 expression oppositely
through binding this region
Results
Analysis of genomic structure and identification
of transcription start site of human KCTD10
To determine the genomic structure of human KCTD10, a full-length cDNA sequence of human KCTD10 was used to search the human genome data-base The results showed that human KCTD10 was mapped to chromosome 12q24.11 region, spanned
21.3 kb and contained five exons Mouse and human KCTD10 share identical organization and exons with similar length (Fig 1A) In the human genome, KCTD10 was arranged in the reverse direc-tion to its neighbor gene – ubiquitin protein ligase E3B (UBE3B, NM_183415) The 5¢-flanking region was very short, only 380 bp between the start the codon of human KCTD10 and 5¢ cDNA end of UBE3B (Fig 1B)
The transcription start site (TSS) of KCTD10 was determined by primer extension assay An antisense primer on the 3¢-end of the first exon was labeled with [32P]ATP[cP], and extended with AMV reverse trans-criptase using total RNAs from HeLa cells as template; simultaneously, sequencing reaction was per-formed using the same primer Nucleotide A, 63 bp upstream of the start codon, was identified as the TSS
by sequence reading (Fig 2)
Identification of the cis-regulatory region and trans-regulatory factors responsible for the promoter activity of human KCTD10 Computer-aided analysis was performed to predict the potential cis-elements regulating KCTD10 expres-sion in the 5¢-flanking region, a 639 bp genomic sequence upstream of the start codon of KCTD10
We did not find any TATA box or CCAAT box around the TSS, but checked out a CpG island with 64.7% GC content throughout the whole 639 bp sequence The 5¢-flanking region of mouse KCTD10 displayed very similar genomic organization to that
of human KCTD10
To characterize potential sequences involved in the regulation of KCTD10 expression, the 5¢-flanking region was amplified, and inserted into the upstream
of pTAL-Luc – a promoter-free luciferase reporter vector A series of 5¢ deletion constructs with a com-mon 3¢-end except for construct P ()609 ⁄ )241) were generated using different primers (Table 1) Luciferase activities of these constructs were assayed in trans-fected HeLa cells As shown in Fig 3, construct P ()609 ⁄ +30) containing the whole 639 bp sequence
Trang 3produced 17.5-fold higher luciferase activity
com-pared with the control pLuc vector Taking this as a
100% base, deletion from )609 to )343 showed a
7.4% increase; construct P ()609 ⁄ )241), in which
271 bp of the 3¢-end was deleted, presented almost
no promoter activity; by contrast, construct P
()241 ⁄ +30) containing this 271 bp of the 3¢-end
showed only a 8.7% increase These results suggested
that the functional promoter region was located in
the 271 bp region of the 3¢-end (from )241 to +30)
Further deletion extended to )203 showed an
28.1% decrease compared with P ()241 ⁄ +30),
whereas deletion to )108 showed an 67.4%
increase compared with P ()203 ⁄ +30) This indicated
that the potential positively-regulating region might
exist from )241 to )203, and the potential
negative-regulating region might exist from )203 to )108
Deletion to )13 sharply decreased luciferase activity
to only 1.68% of P ()609 ⁄ +30) The 5¢-flanking
region contained the functional promoter; within it,
the region from )108 to +30 is essential for the
pro-moter activity
Using tfsearch and matinspector programs,
multiple potential binding sites of transcription
fac-tors were found in the promoter region from )108
to +30 (Fig 1B), including two binding sites for
Sp1, AP-2a and c-myb, and one CACCC box
Mutational analysis revealed that mutation of the upstream Sp1 site (Sp1.1) decreased the promoter activity by 46.9%, whereas mutation of the down-stream AP-2a site (AP-2.2) strongly increased the promoter activity by 88.1% (Fig 4A) However, although other sites were mutated individually, either
no effects (c-myb.2, AP-2.1 and Sp1.2, data not shown) or very modest effects (mutation of the c-myb.1 site decreased the promoter activity by 28.6%, and mutation of CACCC box increased the promoter activity by 27.4%) were observed (Fig 4A) Taken together, Sp1.1 and AP-2.2 were two impor-tant elements related to the regulation of human KCTD10 expression
By aligning the 5¢-flanking region of both human and mouse KCTD10 promoters (human from )144 to )45, mouse from )143 to )44 relative to the start codon), we found that two sequences shared great identities, and Sp1.1 sites were highly conserved (Fig 4B) Although one nucleotide varied from G to T
in the AP-2.2 site (from )77 to )69) of mouse KCTD10 promoter, the sequence (GCCCCCTGC) might also be able to be recognized by AP-2a, and play similar role in the regulation of KCTD10 expression Thus, we proposed that the regulatory mechanisms of KCTD10 might be conserved between human and mouse
A
B
Fig 1 Genomic structure of human and mouse KCTD10 genes, and the 5¢-flanking region sequence of human KCTD10 (A) Genomic organization of human and mouse KCTD10 genes Solid boxes indicate coding region, and open boxes indicate 5¢UTR and 3¢UTR (B) 5¢-Flanking region of human KCTD10 from )609 to +30 KCTD10 was arranged in the reverse direction to its neighboring gene ubiquitin protein ligase E3B (UBE3B, NM_183415); TSS of human KCTD10 is denoted +1, and the start codon (ATG) is indicated by solid box The consen-sus binding sites of the transcription factors with high regulatory activities are boxed, other sites with no or weak regulatory activities are underlined.
Trang 4Binding of Sp1 and AP-2a to the corresponding
sites in the promoter of human KCTD10 in vivo
To confirm that Sp1 and AP-2a bind to the
authen-tic endogenous cis-elements in the KCTD10
pro-moter, we generated a pool of DNA fragments from
HeLa cell lysates using chromatin
immunoprecipita-tion (ChIP) with antibodies against Sp1 or AP-2a
The ChIP-DNA was used as a template for PCR
amplification of the genomic regions containing Sp1
or AP-2a binding elements in the KCTD10
pro-moter, and representative ChIP-PCR results were
shown in Fig 5 The chromatin fragment containing
Sp1- or AP-2a-binding sites in the KCTD10
pro-moter was precipitated by antibodies against Sp1 or
AP-2a but not by control IgG, indicating that
endogenous Sp1 or AP-2a proteins specifically bound
the Sp1 or AP-2a sites, and worked in the basal
state to regulate KCTD10 transcription, respectively
Roles of Sp1 and AP-2a in the regulation of the promoter activity and the expression of human KCTD10
To test the effects of Sp1 and AP-2a binding on KCTD10 promoter activity, we performed a luciferase assay in HeLa and HepG2 cells by overexpression of Sp1 or AP-2a In HeLa cells, co-transfected pCMV-Sp1 increased the luciferase activity of construct P ()241 ⁄ +30) by 58.3%; but only weakly increased the luciferase activity of the Sp1.1 site mutated construct Mt-Sp1.1 (Fig 6A) In AP-2a-deficient HepG2 cells, several reporter constructs containing 5¢-flanking region of KCTD10 such as P ()609 ⁄ +30), P ()108 ⁄ +30) and P ()13 ⁄ +30) presented higher lucifer-ase activities than in AP-2a-normal HeLa cells (Fig 6B); and co-transfected pCMV–AP-2a deceased the luciferase activity of construct P ()241 ⁄ +30) in a dose-dependent manner (Fig 6C) These data sug-gested that Sp1 upregulated, but AP-2a downregul-ated, the promoter activity of human KCTD10 Finally, to determine if the expression changes of AP-2a or Sp1 alter the KCTD10 level, we checked endogenous KCTD10 expression in HeLa cells at the mRNA and protein levels using quantitative real-time RT-PCR and western blot through overexpressing or suppressing AP-2a or Sp1 As shown in Fig 7A, mRNA levels in HeLa cells were measured using quan-titative real-time RT-PCR, and presented as base 10 logarithms For AP-2a, overexpression of AP-2a (A1, purple bar versus yellow bar) led to a 5.6-fold decrease
of KCTD10 at its transcription level (A3, purple bar versus yellow bar, base 10 logarithms); whereas AP-2a knockdown (A1, red bar versus yellow bar) caused a 2.34-fold induction of KCTD10 mRNA level (A3, red bar versus yellow bar, base 10 logarithms) For Sp1, its overexpression (A1, green bar versus yellow bar) resulted in a 12.0-fold increase (A3, green bar versus yellow bar, base 10 logarithms); whereas its knock-down (A1, blue bar versus yellow bar) resulted in a 1.9-fold decrease in KCTD10 mRNA level (A3, blue bar versus yellow bar, base 10 logarithms) Western blot verified these results from quantitative real-time RT-PCR again As shown in Fig 7B–D, whereas AP-2a suppression by RNA interference (RNAi) increased KCTD10 (lane 3 in Fig 7B,D), AP-2a overexpression decreased KCTD10 (lane 2 in Fig 7B,D) However, overexpression of Sp1 increased KCTD 10 (Fig 7C lane 2, Fig 7D lane 4), but silencing of Sp1 decreased KCTD10 (Fig 7C lane 3, Fig 7D lane 5) So we concluded that the expression of KCTD10 could be regulated by Sp1 positively, but by AP-2a negatively
Fig 2 Transcription start site of human KCTD10 Antisense primer
from +21 to +40 was labeled with [32P]ATP[cP] using T4
polynucle-otide kinase, then annealed to total RNAs template from HeLa
cells, and extended with AMV reverse transcriptase DNA
sequence ladder was obtained using the same primer and
sepa-rated on the same gel The extended product is indicated by the
arrow (PE means primer extension).
Trang 5Our data showed that KCTD10 contains neither a
canonical TATA box nor a CCAAT motif in the
pro-moter region, but a cluster of CpG dinucleotides near
to the TSS Although only a single TSS of human
KCTD10 was detected 63 bp upstream of the start
codon, and deletion mutagenesis demonstrated that the
region from )108 to +30 was indispensable for basal
promoter activity, we still could not exclude the
exis-tence of other potential weak initial sites or other weak promoter region which were often found in CpG islands containing and TATA-free promoters [10–12] Typically, GC-rich promoter regions lack TATA or DPE core elements, and are frequently found to be bound with transcription factor Sp1 Sp1 helps to maintain the hypomethylation of CpG islands [13,14], and interacts with some components of the basal tran-scription complex [15,16]; therefore it plays a critical role in the assembly of the transcription start complex
Table 1 Oligonucleotides used in this study.
Fig 3 Function analysis of 5¢-flanking region of human KCTD10 promoter Constructs containing sequentially deleted fragments of human KCTD10 5¢-flanking region (from )609 to +30) were transfected into HeLa cells, luciferase activities were then measured 36 h after trans-fection The region from )108 to +30 is essential for the promoter activity Data (means ± SD) were presented as the percentage of the control, P ( )609 ⁄ +30) Vacant vector pTAL-luc only containing a basal TATA-like promoter was used as experimental control.
Trang 6to selectively activate transcription [13,14,17,18].
In vivo ChIP assays confirmed that Sp1 could
specifi-cally recognize the GC-box element in the proximal
promoter region of KCTD10 Luciferase assays in
HeLa cells indicated that mutation of the Sp1-binding
site decreased the promoter activity by 46.9%,
whereas overexpression of Sp1 increased the promoter
activity by 58.3% The expression of endogenous
KCTD10 was downregulated 1.9-fold by Sp1
silenc-ing, and upregulated 12.0-fold by Sp1 overexpression
Western blot results of KCTD10 were identical to
quantitative real-time RT-PCR Sp1 is an essential
and positive regulator for the transcription of human KCTD10
The binding affinity and transcriptional specificity of Sp1 can be altered by interaction with other cofactors
in the binding sites near to Sp1 recognition motif, such
as C⁄ EBP [19], NF-jB [20], AP-1 [21] or AP-2 [22,23]
In particular, AP-2 binding sites have been found
in most Sp1-dependent promoters AP-2 was a tissue-specific transcription factor, playing critical roles in the regulation of gene expression during mammalian devel-opment, differentiation and carcinogenesis [24–27] It was reported that AP-2 could stimulate AP-2-depen-dent promoter activities in a tissue-specific manner [28], but suppress Sp1-dependent housekeeping pro-moter activities [22,23,29] In this study, we determined the functional role of AP-2a in the regulation of KCTD10 Mutation of the AP-2-binding site increased KCTD10 promoter activity by 88.1%, whereas overex-pression of AP-2a in HepG2 cells inhibited promoter activity in a dose-dependent manner AP-2a silencing increased the expression of endogenous KCTD10 by 2.3-fold, whereas AP-2a overexpression decreased the expression of endogenous KCTD10 by 5.6-fold AP-2a
is a negative regulator for KCTD10
Transcriptional interference, in which overexpression
of one transcription factor resulted in inhibition of another, was often found in the regulation of eukary-otic gene expression The repression of AP-2 on Sp1-dependent transcriptional activity has been explained
in three models: (a) steric hindrance model – AP-2
A
B
Fig 4 Identification of potential binding
sites of transcription factors in the proximal
promoter region (A) Site-directed mutation
analysis of c-myb.1, CACCC-box, Sp1.1 and
AP-2.2 sites in the proximal promoter region
of human KCTD10 Data (means ± SD)
were presented as the percentage versus
the wild-type control P ( )203 ⁄ +30) The
cross (·) indicates the mutated sites.
SREBP-1, sterol regulatory element binding
protein-1 (B) Alignment of the 5¢-flanking
sequences upstream of the start codon of
human and mouse KCTD10.
Fig 5 In vivo ChIP assays of Sp1.1 and AP-2.2 binding sites in the
proximal promoter region of human KCTD10 Mouse monoclonal
anti-Sp1 (lane 2), or rabbit polyclonal anti-(AP-2a) (lane 5), or
nonim-mune mouse and rabbit IgG (lanes 3, 6) were used in ChIP The
DNA fragments derived from AP-2a-specific or Sp1-specific
immu-noprecipitations were amplified using primers flanking the binding
elements of Sp1 or AP-2a A portion of the total input was used as
positive control (lanes 1, 4).
Trang 7represses Sp1-dependent promoter activity by
interfer-ing with the activation of transcription initiation by
Sp1 via specific steric interference machinery [29]; (b)
interaction model – the interaction between Sp1 and AP-2 affects formation of the Sp1–DNA complex [23]; (c) competing model – the AP-2 and Sp1 binding sites overlap, and the AP-2 binding competes with the SP1 binding [22] Our results reveal that the repressive activity of AP-2a on KCTD10 transcription works through inhibition of Sp1 activity It has been pro-posed that the ratio of Sp1 to AP-2a might be respon-sible for the transcriptional state of Sp1-dependent promoters; for example, Chen et al have shown that K3 keratin gene transcription was regulated by the ratio of Sp-1 to AP-2 in differentiating rabbit corneal epithelial cells [29] In our case, even though there were similar levels of Sp1 in both HeLa and HepG2 cells, KCTD10 promoter activity is significantly lower in HeLa cells than in HepG2 cells; this is probably due
to the AP-2 deficiency When AP-2a was overexpressed
in HepG2 cells, KCTD10 promoter activity decreased
in a dose-dependent manner However, AP-2a silenc-ing increased KCTD10 transcription, whereas AP-2a overexpression repressed KCTD10 transcription in HeLa cells It is possible that the relative ratio of Sp1
to AP-2a in a particular cell environment determined the activation or repression of human KCTD10 Our previous study demonstrated that mouse PDIP1 could also be regulated by Sp1 and AP-2, and the reg-ulating sites were conserved in human, mouse and rat [30], which implied that the PDIP1 gene family might have similar regulating mechanisms in different species
It has been reported that Sp1 binding to the promoters were critical for the activation of TNF-a- stimulated ADAM17[31] and MCP-1 genes [32]; however, we did not detect significant changes in KCTD10 expression
in HeLa cells after TNF-a induction (data not shown) This suggested that the isolated 5¢-flanking region of KCTD10 might not contain a TNF-a responsive element Although our results confirmed the roles of two transcription factors, Sp1 and AP-2a, in the
A
B
C
Fig 6 Effects of Sp1 and AP-2a expression level on the promoter activity of human KCTD10 (A) The relative luciferase activities of P ( )241 ⁄ +30) and Mt-Sp1.1 mutant were examined in pCMV–Sp1-transfected HeLa cells pCMV–myc was used to equilibrate the plasmid quantity, and pCMV–LacZ was used as a transfection effi-ciency control Data (means ± SD) were presented as the percent-ages of wild-type promoter control (B) Constructs P ( )609 ⁄ +30), P ( )108 ⁄ +30), P ()13 ⁄ +30) and pTAL-luc control were transfected into HeLa or HepG2 cells, respectively Luciferase activities were presented as the percentages of pTAL-luc control (C) The relative luciferase activities of P ( )241 ⁄ +30) were inhibited by AP-2a over-expression in a dose-dependent manner in pCMV-AP-2a
transfect-ed HepG2 cells pCMV–myc was ustransfect-ed to equilibrate the plasmid quantity, and pCMV–LacZ was used as a transfection efficiency control.
Trang 8regulation of KCTD10 expression, other regulatory
proteins involved in the transcription control of
KCTD10still need to be further investigated
Experimental procedures
Mapping TSS by primer extension The TSS of human KCTD10 was determined using primer extension kit (Promega, Madison, WI, USA) according to the manufacturer’s instruction Briefly, an anti-sense primer (5¢-TCTCCAAACCCGGACTGAGA-3¢) corresponding to the position from +21 to +40 was labeled with [32P]ATP[cP] using T4 polynucleotide kinase, and purified by precipitation
in 75% ethanol The labeled primer was incubated with
25 lg of total RNA from HeLa cells at 62C for 30 min, and then extended with AMV reverse transcriptase at 42C for 1 h; sequencing reaction was further performed using CycleReader DNA Sequencing kit (Invitrogen, San Diego,
CA, USA) with the same labeled primer The products of primer extension and sequencing reaction were resolved on 6% denaturing polyacrylamide sequencing gel
Computer analysis of KCTD10 promoter structures, cloning of the promoter fragment, construction of deletion and point-mutated vectors Promoter searching was performed using proscan (http:// www-bimas.cit.nih.gov) and promoterinspector (http:// www.genomatix.de) programs Transcription factor binding sites in the 5¢-flanking region of human KCTD10 were pre-dicted using tfsearch (http://www.cbrc.jp/research/db/ TFSEARCH.html) CpG island in this region was analyzed (http://cpgislands.usc.edu) using the criteria of CG percent-ages over 50%, calculated or expected CpG ratio over 0.6 and a minimal length of 200 bases
The human genomic chromosome 12 clone (NT_ 009775.15) containing KCTD10 was identified by searching the human genome using full-length KCTD10 cDNA sequence A 639 bp genome fragment upstream of the start codon (ATG) and other five 5¢-deletion mutants were obtained by PCR from human genomic DNA using the 3¢-end primer pLuc-KCTD10-R and the upstream 5¢-end primers F1 to F6 (Table 1), and subcloned into the KpnI⁄ HindIII sites of pTAL-Luc vector (Clontech, Mountain View, CA, USA) A 3¢-deletion mutant was constructed using the primers P ()241 ⁄ +30)-F3 and P ()609 ⁄ )241)-R Site-directed mutants derived from the P ()203 ⁄ +30) construct were generated by overlapping extension PCR as described previously [10] In brief, in the first round, two PCR were performed in parallel using construct P ()203 ⁄ +30) as a template: one with a wild-type forward primer P ()203 ⁄ +30)-F4 and a reverse mutated primer, the other with a forward mutated primer (complementary to the reverse mutated primer) and a wild-type reverse primer PLuc–KCTD10-R; in the second round, equal molar mixture of the two PCR products was used as template,
P ()203 ⁄ +30)-F4 and pLuc–KCTD10-R were used as primers The final PCR products were similarly subcloned
A
B
C
D
Fig 7 Endogenous expression change in human KCTD10 after
overexpressing, or suppressing Sp1 and AP-2a (A) mRNA levels in
HeLa cells were measured using quantitative real-time RT-PCR,
and presented as base 10 logarithms A1, mRNA levels of AP-2a
after overexpressing Ap-2a, or suppressing AP-2a by RNA
interfer-ence; A2, mRNA levels of SP1 after overexpressing Sp1, or
sup-pressing Sp1 by RNA interference; A3, mRNA levels of human
KCTD10 after overexpressing Ap-2a or Sp1, or suppressing AP-2a
or Sp1 by RNA interference mRNA levels in HeLa cells transfected
with Myc vector were used as negative controls In A3, folds of
human KCTD10 mRNA levels versus negative control were
calcu-lated All experiments were run three times (B–D) The protein
expressions of AP-2a, Sp1 and KCTD10 after overexpressing Ap-2a
or Sp1, or suppressing AP-2a or Sp1 by RNA interference in HeLa
cells were examined using western blotting b-Actin was used as
internal control; Si-NC represents scramble siRNA oligos which can
not knockdown AP-2a, SP-1 and KCTD10.
Trang 9into pTAL–luc vector All constructs were confirmed by
DNA sequencing
Luciferase reporter assay
HeLa and HepG2 cells were cultured in DMEM
(Invitro-gen) supplemented with 10% fetal bovine serum, 2 mm
l-glutamine, 100 UÆmL)1 penicillin and 100 lgÆmL)1
strep-tomycin at 37C in a 5% CO2incubator When cells reach
90% confluence in 24-well plate, the culture medium was
replaced with serum- and antibiotic-free medium 3 h before
transfection; then 0.5 lg of luciferase reporter plasmids
were co-transfected with 0.3 lg of b-galactosidase
expres-sion vector pCMV-LacZ using Lipofectamine 2000
Reagent (Invitrogen); 6 h after transfection, cells were
washed, and fresh medium supplemented with serum and
antibiotics was put back again; cell lysates were collected
36 h after transfection and assayed for luciciferase activity
using the Luciferase Assay System (Promega)
Mammalian expression vectors of human Sp1
and AP-2a
The human Sp1 coding sequence was amplified from HeLa
first-strand cDNA using forward primer with EcoRI site
(underlined) and reverse primer with SalI site in 5¢-end
(for-ward primer: 5¢-AGAATTCCTGCCACCATGAGCGAC
CA-3¢, reverse primer: 5¢-CGTCGACTG ATCTCAGAA
GCCATTTTGC-3¢) The PCR product was subcloned into
pCMV–HA (Clontech) at EcoRI⁄ SalI sites The human
AP-2a coding sequence was amplified from a human brain
cDNA library using the primer shown in Table 1, and
sub-cloned into pCMV–HA at EcoRI⁄ XhoI sites Plasmids
pCMV–Sp1 and pCMV–AP-2a were confirmed by
sequenc-ing, and used to overexpress Sp1 and AP-2a in mammalian
cells All expressed proteins were confirmed by SDS–PAGE
and western blotting
ChIP assay
Sequence-specific DNA binding activities of Sp1 and AP-2a
to KCTD10 promoter were confirmed by in vivo ChIP
assays EZ ChIP Kit (Upstate, Temecula, CA, USA) was
used following the manufacturer’s instruction Briefly, cells
were cross-linked for 10 min in 1.6% formaldehyde
solu-tion; for every 2 mg of protein extracts, 10 lg of mouse IgG
bound to protein A–Sepharose was used for 1 h pre-clear
with shaking at 4C; protein extracts were then
immuno-precipitated with 2 lg of rabbit polyclonal anti-(AP-2a) or
2 lg of mouse monoclonal anti-Sp1 (Santa Cruz Biotech,
Santa Cruz, CA, USA) by rotating overnight at 4C To
generate the pool of DNA fragments, protein extracts from
10 cm plate culture of HeLa cells were immunoprecipitated;
the immunocomplexes were then washed and eluted; after
reversion of cross-link, the DNA fragments were purified using spin columns The PCR primers for ChIP assay (shown in Table 1) were designed to flank the Sp1 and AP-2a sites in KCTD10 promoter
Real-time quantitative RT-PCR and western blot Specific small interfering RNA (siRNA) against AP-2a (5¢-GCUCCACCUCGAAGUACAATT-3¢) and Sp1 (5¢-NN AGCGCUUCAUGAGGAGUGA-3¢) [12] were synthesized from Invitrogen to suppress the endogenous expression of Sp1 and AP-2a; pCMV–Sp1 and pCMV–AP-2a were used
to overexpress Sp1 and AP-2a The cultured cells were transfected using Lipofectamine 2000 according to the manufacturer’s instruction; 24 h later after transfection, total RNAs were extracted from 107 cells with TRIzol reagent (Invitrogen); RT-PCR was carried out using a two-step strategy following the manufacturer’s manual (Pro-mega) The primers for quantitative real-time RT-PCR of KCTD10 were shown in Table 1, and b-actin was used for internal normalization In parallel, nuclear extracts were prepared from the transfected cells; after 5 min heating and separation on 12.5% denaturing polyacrylamide gel, pro-teins were transferred onto nitrocellulose membranes, and then analyzed by western blot using rabbit polyclonal serum against AP-2a or Sp1 (Santa Cruz Biotech)
Acknowledgements
This work was supported in part by the National Nat-ural Science Foundation of China (No 30771082, 30571005), the 973 project of Ministry of Science and Technology of China (No 2005CB522505, 2006CB943506), the Hunan Provincial Natural Science Foundation of China (No 08JJ3082), Project of Hunan Science and Technology Commission: run foundation of large-scale precision instrument and management of experiment animals (2060599), Project of Changsha Science and Technology Department (K0803107-21) Changsha, the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Educa-tion of China (No 705041), the Provincial Science & Technology Department of Hunan (05FJ4016), and pro-vincial Education Department of Hunan (No 05C391, 07C563)
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