modulation of transcription of the mouse testis- andKong-Bung Choo1,2,3, Min-Chuan Hsu1,2, Yao-Hui Tsai1,4, Wan-Yi Lin1and Chiu-Jung Huang4 1 Department of Medical Research and Education
Trang 1modulation of transcription of the mouse testis- and
Kong-Bung Choo1,2,3, Min-Chuan Hsu1,2, Yao-Hui Tsai1,4, Wan-Yi Lin1and Chiu-Jung Huang4
1 Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan
2 Department of Biotechnology and Laboratory Science in Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
3 Institute of Clinical Medicine, National Yang Ming University, Taipei, Taiwan
4 Department of Animal Science and Graduate Institute of Biotechnology, School of Agriculture, Chinese Culture University, Yangmingshan, Taipei, Taiwan
Introduction
We have previously reported two tripartite motif
(TRIM)⁄ RING-Box-coiled coil (RBCC) protein
genes – Rnf33⁄ Trim60 and Rnf35 ⁄ Trim61 – that are
temporally expressed in the egg and in the pre-implan-tation embryo of the mouse; both genes are silenced at the blastocyst stage before placental implantation and
Keywords
nuclear factor-kappa B (NF-jB); p65 and p50
proteins; Rnf33; testis-specific gene
expression; tumor necrosis factor-alfa
(TNF-a)
Correspondence
Chiu-Jung Huang, PhD, Associate Professor,
Department of Animal Science and
Graduate Institute of Biotechnology, School
of Agriculture, Chinese Culture University,
55, Hwa-Kang Road, Yangmingshan,
Taipei 111, Taiwan
Fax: +886 2 28613100
Tel: +886 2 28610511 ext 31231
E-mail: hqr2@faculty.pccu.edu.tw
(Received 29 July 2010, revised 15 November
2010, accepted 24 December 2010)
doi:10.1111/j.1742-4658.2010.08002.x
We have previously reported a mouse Rnf33⁄ Trim60 gene that is temporally expressed in the pre-implantation embryo The Rnf33 structural gene is com-posed of a short noncoding exon 1 and an intronless coding exon 2 In the present work, Rnf33 was shown to be expressed in the mouse testis and in the testicular cell lines TM3 and TM4 To elucidate Rnf33 transcriptional modu-lation, a 2.5-kb Rnf33 sequence, inclusive of the upstream regulatory region, exon 1 and the associated intronic sequence, was dissected in transient trans-fection and luciferase assays An initiator and an atypical TATA-box were shown to act as the core promoter elements of the gene Deletion and muta-genesis of the 2.5-kb sequence in luciferase constructs further demonstrated that an intronic and palindromic kappa B (jB) sequence was an important ciselement targeted by the nuclear factor-jB (NF-jB) subunits p65⁄ RELA and p50⁄ NFjB1, and also through modulation by tumor necrosis factor a Transcriptional up-regulation of Rnf33 by NF-jB and tumor necrosis
factor-a wfactor-as directly demonstrfactor-ated in TM3 factor-and TM4 cells by refactor-al-time PCR qufactor-anti- quanti-fication of the Rnf33 mRNA levels Small interfering RNA knockdown of p65 and p50 confirmed Rnf33 down-regulation by p65⁄ p50 Spermatogenesis
is regulated by a wide range of stimuli, including NF-jB, which, in turn, is regulated by other signals Hence, demonstration of NF-jB-regulated Rnf33 expression in testicular cells, particularly in Sertoli cells, implicates functional involvement of the putative RNF33 protein in spermatogenesis through association of the RNF33 protein with the microtubule via interaction with kinesin motor proteins, as previously demonstrated [Huang et al., submitted]
Abbreviations
AR, androgen receptor; aTATA, atypical TATA-box; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIF-1a, inducible factor 1a; HRE, hypoxia-response element; IKK, IjB kinase; Inr, initiator; KIF3A⁄ KIF3B, kinesin-2 family members 3A and 3B; NF-jB, nuclear factor-kappa B; RBCC, RING-Box-coiled coil; SF, serum-free; siRNA, small interfering RNA; SV40, simian virus 40; TFBSs, transcription factor-binding sites; TNF-a, tumor necrosis factor a; TRIM, tripartitate motif; jB, kappa B.
Trang 2remain silenced throughout the remaining stages of
embryonic development [1,2] The Rnf33 gene is
located 11.5 kb downstream of Rnf35; both Rnf33 and
Rnf35 are intronless in the coding sequences but each
gene is associated with a short noncoding exon 1 and
therefore with a single short intron of about 2.2 and
3.3 kb in size, respectively (Fig 1) [1,2]
Transcriptional regulation of the upstream Rnf35
gene has been closely examined [3,4] The Rnf35
pro-moter is TATA-less but utilizes an initiator (Inr)
sequence as the core promoter element Two
transcrip-tion factors have been identified that participate in
Rnf35 expression: the ubiquitous positive regulator
nuclear factor Y (NF-Y) that binds to Y-box motifs in
the upstream regulatory sequence, and the repressor
CCAAT-displacement protein (CDP) that targets a cis
sequence in exon 1 [3,4] We have also shown, in a
pre-vious work, that the bulk of the Rnf33 transcripts in
the pre-implantation embryo are initiated from a
major promoter, designated P1 in Fig 1, located
immediately upstream of the major transcription start
site [1] Other weak Rnf33 transcription start sites have
also been identified in the early embryo, including
one that exploits the single major promoter of the
upstream Rnf35 gene, indicating occasional erratic
co-transcription of the Rnf35 and Rnf33 genes in early
development (Fig 1) [1] Rnf33 encodes a putative
TRIM protein composed of a typical RBCC and a
B30.2 domain; TRIM⁄ RBCC proteins have been
implicated in development, cell growth, differentiation
and other biological functions [5] In another study, we
have shown that RNF33 interacts with the kinesin-2
family members 3A (KIF3A) and 3B (KIF3B) motor
proteins in heterodimeric form, possibly contributing
to the KIF3A⁄ KIF3B-dependent cargo-mobilization
process along the microtubule in the pre-implantation
embryo and in the testis [Huang, Huang, Chang, Hsu,
Lin & Choo, submitted] Other TRIM proteins that
are associated with the microtubule have been shown
[6,7]
In this work, we aimed to further elucidate
tran-scriptional regulation of the Rnf33 retrogene First of
all we reported expression of Rnf33 in the mouse testis and testicular Sertoli and Leydig cell lines We identified a positive-acting kappa B (jB) element that was located in the single intron of the Rnf33 gene adjacent to exon 1; the intronic jB was targeted by the p65⁄ RelA and p50 ⁄ NFjB1 nuclear factor-jB (NF-jB) subunits, and the jB-dependent transcrip-tion was also modulated by tumor necrosis factor alpha (TNF-a) p65⁄ p50 transcriptional modulation
of Rnf33 in Sertoli cells was further confirmed by small interfering RNA (siRNA) knockdown of p65⁄ p50 expression, which resulted in Rnf33 down-regulation Our findings suggest possible functional involvement of the putative RNF33 protein in sper-matogenesis in Sertoli cells under the regulation of NF-jB
Results
Testicular expression of Rnf33 The Rnf33 gene has previously been shown to be temporally expressed only in the mouse pre-implanta-tion-stage embryo and not in the major tissues tested [1]; this finding is supported by the approximate expression profile based on the expressed sequence tag (EST) database of GenBank (data not shown) However, in this study we detected Rnf33 mRNA in the testis and in two mouse testicular cell lines: TM3 and TM4 (Fig 2) TM3 and TM4 are nontumorigenic epithelial cell lines derived from mouse testicular Leydig and Sertoli cells, respectively [8] Leydig cells are interstitial cells located adjacent to the seminifer-ous tubules in the testicle; the cells synthesize and secrete androgens in response to stimulation with the pituitary luteinizing hormone Sertoli cells form part
of the seminiferous tubule, and the main function of the cells is to nurture the developing sperm cells through spermatogenesis The expression of Rnf33 mRNA in TM3 and TM4 cells suggests that Rnf33 transcription may occur in both the Leydig and Ser-toli cells of the testis
P1
CDP NF-Y
Rnf35 Rnf33 TSS’s Rnf33
Inr
Rnf35
promoter
1 kb
Fig 1 Map of the Rnf35 and Rnf33 genes The relative map positions of the intronless genes (boxes) are as established previously [1,2] Thick horizontal bars denote untranslated regions; filled arrowheads with solid lines indicate major RNA start sites; arrowheads with dashed lines denote minor Rnf33 RNA start sites used in pre-implantation embryos; and slanting dashed lines represent splicing events P1 denotes the major Rnf33 RNA start sites used in both the pre-implantation embryo and in the testis, as reported in this work In the Rnf35 promoter,
an Inr element, two binding sites for nuclear factor Y (NF-Y) and one binding site for the CCAAT-displacement protein (CDP) are shown, as reported previously [3,4] TSS’s, transcriptional start sites.
Trang 3Identification of the Rnf33 core promoter
elements and a cis-acting transcriptional
sequence in the intron
To elucidate cis-acting transcriptional elements of
Rnf33, a 2560-bp DNA sequence, designated as F1,
which included 1 kb of upstream regulatory sequence,
the 245-bp noncoding exon 1 and 1 kb of the flanking
intronic sequence (sequence)1287 to +1271, Fig 3A),
was cloned into the promoter-less luciferase vector,
pGL3-Basic, for use in transient transfection and
lucif-erase assays in the permissive CHO-K1 cell line, as
previously described for the Rnf35 gene [3,4] In the
F1 sequence, a putative atypical TATA-box (aTATA)
and a putative Inr element could be discerned
(Fig 3A; sequence on top) For analysis of their roles
in transcription, aTATA was deleted in construct
F1DaT and Inr was mutated in construct F1mutI; both
the aTATA deletion and the Inr mutation were
included in the double mutant F1DaT⁄ mutI (Fig 3A,
left-hand panel) In transfection and luciferase assays,
a reduction of 35% or 25% in luciferase activity
was observed in cells transfected with F1DaT or
F1mutI, respectively, relative to the wild-type F1
luci-ferase activity (Fig 3A, right-hand panel) In cells
transfected with the double mutant, the luciferase
activity was further decreased to 50%, suggesting a
combined contribution of aTATA and Inr as the
Rnf33 core promoter elements Interestingly, mutating
both aTATA and Inr failed to completely ablate
tran-scriptional activities, indicating the presence of other
important transcriptional cis sequences in the
neigh-borhood This was supported by the detection of only
10% transcriptional suppression following
transfec-tion with the construct R1, in which the upstream
sequence is deleted but the two core promoter elements
and downstream sequences are retained When both
aTATA and Inr were mutated in R1 in the R1DaT⁄
mutI construct, a residual luciferase activity of 35%
was detected, indicating the presence of key
transcrip-tional regulatory elements in the retained exon 1 and
possibly also in the intron sequence This hypothesis gained support when progressive deletions of the in-tronic sequence of F1 from the 3¢ end in constructs F2 and F3 led to a progressive reduction in transcriptional activities The first 3¢ segment deleted in the intron sequence (designated R4) in the construct F2 resulted
in the abolishment of 80% of the relative luciferase activity, despite the presence of exon 1 and the upstream sequence (Fig 3A) A further deletion in construct F3 confirmed the importance of R4, albeit with possible further contribution outside the R4 sequence When one to three copies of the 305-bp R4 sequence were cloned in either the forward or reverse orientations in front of the constitutive simian virus 40 (SV40) promoter, or 3¢ to the luciferase gene, in the pGL3-promoter reporter plasmid, luciferase assays showed that R4, in a single copy in either orientation, up-regulated SV40 promoter activities when placed upstream or downstream of the luciferase gene, indi-cating enhancer-like functions (Fig 3B, US-R4F and DS-R4F) Furthermore, the enhanced transcriptional activities were additive: up to three- or fivefold up-reg-ulation in the SV40 promoter activities was achieved when three copies of R4 were placed downstream of the luciferase gene and in the forward or reverse orien-tation, respectively (Fig 3B, DS-3R4F and DS-3R4R) Taken together, our data indicate that an aTATA and
an initiator act as the core promoter elements in Rnf33 transcription in the presence of crucial, positive cis-act-ing transcriptional element(s) in the R4 sequence resid-ing in the only intron of Rnf33
Identification of a jB element as the crucial cis regulatory sequence
To further dissect the transcriptional contribution, the 305-bp R4 was arbitrarily divided into three regions, approximately equal in size, for luciferase assays Puta-tive transcription factor-binding sites (TFBSs) were also identified by bioinformatics analysis (Fig 4A) The R4-1 section was found to contain a jB element
in the sequence 5¢-GGGAATTCCC-3¢, which is the binding site for nuclear factor-jB (NF-jB), a putative hypoxia-response element (HRE) in the sequence 5¢-ACGTG-3¢ that is targeted by hypoxia-induced fac-tor 1a (HIF-1a) and a putative binding site for the GATA transcription factor in the reverse orientation [9,10] No putative TFBSs were discernible in R4-2 In R4-3, an N-box and two E-box motifs were predicted
To delineate the possible contribution of these pre-dicted TFBSs, the three R4 subsections were either retained or deleted individually or in different combi-nations from the F1 construct for luciferase assays
Te T3M TM4 Li
Rnf33
ββ-actin
Fig 2 Testicular expression of Rnf33 RNA samples were
pre-pared from the mouse testis (Te) and from the testicular cell lines
TM3 and TM4 for use in RT-PCR analysis with Rnf33-specific
prim-ers Liver (Li) was included as a negative control, and b-actin was
used as a PCR control.
Trang 4(Fig 4A, left-hand panel) Deletion of R4-2 or R4-3
alone (constructs F1R4-1⁄ 3 and F1R4-1 ⁄ 2) did not
appreciably affect luciferase activities relative to that
of the parental F1 construct (Fig 4A) However,
simultaneous deletion of both R4-2 and R4-3
(con-struct F1R4-1) resulted in an increase, of 50%, in
luciferase activity, suggesting the possible presence of negative regulator(s) in the deleted sequences On the other hand, when R4-1 alone was deleted in construct F1R4-2⁄ 3, the relative luciferase activities were almost abrogated Furthermore, deletion of R4-1, in combination with R4-2 or R4-3 deletion in constructs
A
B
Fig 3 Identification of the core promoter elements and an intronic cis-acting transcriptional regulatory sequence of Rnf33 (A) Identification
of the core promoter elements In the experiments, mutation and deletion luciferase constructs were derived from the F1 fragment that con-tained the upstream regulation region, exon 1 and 1 kb of the Rnf33 intron; the sequences were cloned in front of the promoter-less lucifer-ase gene of pGL-Basic In the sequence display at the top, the putative atypical TATA-box (denoted as aT) and the initiator (Inr) are boxed In the aT deletion mutant (F1DaT) constructs, six nucleotides (doubly underscored), including aT, were deleted (deletion denoted by D); in the Inr mutant (F1mutI) constructs, a four-nucleotide mutation (indicated by downward-pointing arrows and the substituted nucleotides) was introduced (the mutated sites are shown by crosses) In the R1 construct, the upstream regulatory sequence was deleted but aT and Inr were preserved; and in R1DaT ⁄ mutI, the dT was deleted and Inr was mutated F2 and F3 constructs carried three terminal serial deletions
of the intronic sequence of F1, as indicated Transfection and luciferase assays were performed in CHO-K1 cells The data shown are from three independent experiments Relative luciferase activities (RLU) were calculated by arbitrarily setting the luciferase activity of the wild-type F1 construct as 10 The R4 sequence, identified as harboring cis-acting activities (see the text), is shown (B) Confirmation of positive cis transcriptional activities of R4 One or more copies of R4 (thick open arrows) were inserted upstream or downstream of the SV40 promoter (SV Pr, hatched boxes) of the pGL2-promoter vector in either the same (rightward-pointing) or reverse (leftward-pointing) orienta-tion as the luciferase (Luc) gene, as displayed The constructs were individually transfected into CHO-K1 cells for luciferase assays The lucif-erase activity of the parental pGL3 promoter was arbitrarily set as 1 for computation of the RLUs of other constructs In both (A) and (B), data were subjected to the Student’s t-test; *P < 0.05; **P < 0.01 relative to the controls.
Trang 5F1R4-3 or F1R4-2, partially restored transcriptional
activity, consistent with the supposition of the presence
of negative cis regulator element(s) in R4-2 or R4-3, as
described above Hence, deletion analysis further
mapped the presence of positive cis-acting
transcrip-tional element(s) to the 93-bp R4-1 sequence
To investigate the contribution of the three
dis-cerned putative TFBSs in R4-1 to Rnf33
transcrip-tional modulation, these sites were mutated
individually or in combination with one another, and
transfection and luciferase assays were carried out
(Fig 4B) When the HRE was mutated in construct F1MutH, a reduction in luciferase activity of 30% was observed Moreover, mutation of the jB element
in construct F1MutjB led to a reduction of 70% in luciferase activity When the double mutant F1MutjB⁄ H was similarly assayed, the reduction in luciferase activity was not additive but remained at
70%, reflecting the dominant role of jB However, cross-talk between NF-jB and HIF-1 in Rnf33 tran-scription cannot be ruled out because HIF-1a is also a target gene of NF-jB [11,12] On the other hand,
A
B
C
Fig 4 Association of R4 transcriptional
activity with a jB element (A) Further
map-ping of transcriptional activities to a
subsec-tion (R4-1) of R4 The R4 sequence was
arbitrarily divided into three sections – R4-1
to R4-3 – of approximately equal length In
R4-1, the discerned putative TFBSs are jB,
HRE and GATA (denoted by vertical bars); in
R4-2, no TFBSs were identified; in R4-3, an
N-box (N) and two E-boxes (E) were
detected The R4-1 to R4-3 subsections
were retained or deleted individually, or in
different combinations, from the parental F1
sequence (see Fig 3A) In the panel of
con-structs displayed on the left, the R4
seg-ment in F1 is magnified for clarity by
omitting the sequence between exon 1 and
R4 (denoted by the slanting double-break
symbols in the construct displays) (B)
Identification of jB as the major cis
tran-scriptional element in R4-1 The R4-1
sequence is shown at the top; the predicted
TFBSs are boxed; and mutations (denoted
by crosses) that were introduced into the
luciferase constructs are indicated by
down-ward-pointing arrows (C) Confirmation of
positive transcriptional activity of the jB
element In R4-1(jB)4, four jB copies were
cloned upstream of the SV40 promoter (SV
Pr, hatched boxes) of the pGL3 promoter.
Construct US-R4F that carried full-length R4
(see Fig 3B) was included for comparison.
RLU, relative luciferase activities.
Trang 6mutating the putative GATA-binding site (construct
F1MutG) had no discernible effects on transcriptional
activity, ruling out a role of the putative
GATA-bind-ing site in transcription This conclusion is further
supported by the assay of the triple mutant
F1MutjB⁄ H ⁄ G that yielded luciferase activities similar
to that obtained with the jB-only mutant construct
To confirm the contribution of jB to transcription,
four copies of a 12-mer TGGGAATTCCCC sequence,
which included the jB sequence (underlined), were
placed at the 5¢ end of the SV40 promoter of the
pGL3-promoter vector to generate construct R4-1(jB)4
for luciferase assays (Fig 4C) While insertion of the
single-copy jB-containing R4 sequence resulted in a
1.5-fold increase in the SV40 promoter activity, the
presence of four copies of the 12-mer jB sequence
resulted in a significant eightfold increase in promoter
activity relative to the parental plasmid (Fig 4C)
Pro-moter-activity analysis in luciferase assays firmly
estab-lished that the discerned jB element in R4-1 is the
primary cis-acting positive regulatory element, while
the putative HRE sequence may play a secondary role
in Rnf33 transcriptional regulation
The Rnf33 jB element is targeted by the NF-jB
proteins p50 and p65
It has been well established that jB sequences are
tar-geted by the abundantly expressed NF-jB [13–15]
Involvement of the NF-jB in gene regulation in the
testis has also been described [16–20] Among the five
NF-jB proteins, p50⁄ NFjB1 and p65 ⁄ RELA have
clearly been shown to be the major NF-jB proteins
expressed specifically in the testis [21–23] Expression
of p50 and p65 was confirmed in the testis and
estab-lished in the TM3 and TM4 testicular cell lines by
RT-PCR and western blot analyses (Fig 5) It is noted
in the western blots that while the p50 and p65 levels
were relatively constant in the testis, in the two
testicu-lar cell lines and in the control liver tissue, p65 levels
were found to be more than threefold higher in TM3
and TM4 cells than in the testis and liver tissues
(Fig 5B)
To determine p50 and p65 targeting of the R4-1 jB
site, an electrophoretic mobility shift assay (EMSA)
was performed In the presence of nuclear extracts
pre-pared from the TM3 and TM4 cells, a protein-induced
band shift was observed (Fig 6A, lanes 2 and 7,
arrowhead) In the presence of increasing amounts of
the unlabeled wild-type probe sequence, the shifted
band was effectively competed out (Fig 6A, lanes 3,
4, 8 and 9) A jB mutant oligonucleotide, however,
had little effect on the observed band shift (Fig 6A,
lanes 5, 6, 10 and 11) The identity of the jB-bound protein was further established in supershift assays (Fig 6B) On addition of an anti-p65 serum, the pro-tein-induced bands in both TM3 and TM4 cells were obliterated, indicating specific p65 targeting (Fig 6B, lanes 3 and 6); in the experiments, the supershifted bands were not apparent, as previously reported in similar assays in testicular cells [20] However, addition
of an anti-p50 serum did not seem to appreciably affect the protein-shifted band in both TM3 and TM4 cells (Fig 6B, lanes 2 and 5) In in vivo p65-binding assays carried out by chromatin immunoprecipitation (ChIP), the anti-p65 and -p50 sera both yielded vari-ous intensities of Rnf33-specific PCR bands from TM3 and TM4 cells and the testis, but not from the liver (which does not express Rnf33) (Fig 6C) In the mock experiment in which the antibody treatment was omit-ted, or in the case in which a pre-immune antiserum was used, no specific PCR products were detected Taken together, EMSA and ChIP assays indicate that the NF-jB subunit proteins p65, and possibly p50, target the intronic jB site of Rnf33, resulting in tran-scriptional activation of Rnf33 in the testis The p65 protein seems to be preferred over p50 in targeting the Rnf33 jB site, and the protein–target site interactions also appear to be weak
To further verify the specificity of NF-jB transcrip-tional modulation of Rnf33 expression, the expression
of p65 or p50 was knocked down by double-stranded siRNA in TM4 cells (Fig 7) When TM4 cells were
Te TM3 TM4 Li
p50
p65
p50
p65
β-actin
Te TM3 TM4 Li
RL: 1 3.16 3.05 0.74 RL: 1 0.78 0.76 0.83
A
B
Fig 5 Expression of the p65 and p50 NF-jB subunit proteins in testicular cells (A) RT-PCR detection of p50 and p65 transcripts in the testis (Te) and in TM3 and TM4 cells (B) Western blot analysis
of the p50 and p65 proteins The relative protein levels (RL) were computed by normalizing with the b-actin level and were calculated relative to the level of the testis set as 1 Liver (Li) was included as
a control in the analyses.
Trang 7transfected with the p65 siRNA, the relative mRNA
level of p65, as quantified by real-time RT-PCR, was
significantly reduced to 48% of that of nonspecific
siR-NA-transfected cells but the relative p50 mRNA level
was unaffected (Fig 7A) Likewise, transfection with
p50 siRNA resulted in a significant reduction (by
50%) of the p50 transcript levels but not of the p65
transcript levels Effective knockdown of p65 or p50
by the respective siRNA was supported by western
blot analysis, showing a reduction in the p65 and p50
protein levels of approximately 50% and 33%,
respec-tively, in the transfected cells (Fig 7B) When p65 was
knocked down by siRNA, the mRNA level of Rnf33
was significantly reduced to 47.5% of that of the
non-specific control (Fig 7A, Rnf33 panel) However, p50
knockdown did not have any significant effect on the
Rnf33 mRNA level Likewise, when transfected with
the p65 siRNA, the RNF33 protein level was reduced
by approximately 33% relative to the cells transfected
with nonspecific siRNA, but transfection with p50
siR-NA did not result in appreciable reduction of RNF33
protein (Fig 7B, RNF33 panel) The results of the
siRNA experiments clearly support p65-modulated
Rnf33expression and indicate that p50 seems to play a
lesser role than p65 in Rnf33 expression Taken
together, our data demonstrate that Rnf33
transcrip-tion is modulated by the NF-jB p65 protein, probably
in the form of the more ubiquitous p65–p50
hetero-dimer, and possibly also in the p65–p65 homodimeric
form
Rnf33 expression is up-regulated by TNF-a or p50⁄ p65 overexpression via the jB element
To further determine if the observed jB modulation of Rnf33 transcription at the R4-1 jB site was TNF-a dependent, the testicular TM3 and TM4 cells were transfected with the jB-containing luciferase construct (F1), with construct F1R4-2⁄ 3 (from which the jB-containing R4-1 segment had been deleted) or with the
jB mutant construct F1MutkB (see Fig 4 for con-structs), and the transfected cells were treated with TNF-a before luciferase assays were performed The results showed a 50%, significant, increase in luciferase activity in the presence of TNF-a in F1-transfected TM3 cells, and a fourfold, significantly higher lucifer-ase activity in F1-tranfected TM4 cells relative to the untreated cells (Fig 8A) Consistent with previous assays in CHO-K1 cells, luciferase activities were negli-gible or were significantly lower when jB site deletion
or mutated constructs were assayed in both TM3 and TM4 cells, and TNF-a did not elicit discernible effects
on the luciferase activity (Fig 8A) Hence, Rnf33 pro-moter activation in testicular cells is modulated by TNF-a and the modulation is dependent on the pres-ence of the jB site
To test if the jB site is targeted by homodimeric or heterodimeric p50 and p65 proteins, p50 and⁄ or p65 overexpression plasmids were transiently co-transfected with the jB-containing F1R4-1 construct or with the jB mutant construct F1MutkB (see Fig 4 for
Competitor:
–
1 2 3 4 5 6 7 8 9 10 11
ns ns ns
p50:
p65:
– – + – +
–
Testis
TM4 TM3
Liver
InputMock a-p65a-p50Pre
Fig 6 Targeting of the R4-1 jB element by p65 and p50 (A) Electrophoretic mobility shift assay (EMSA) using a jB probe and nuclear extracts (NE) prepared from the mouse testicular cell lines, TM3 and TM4 The open arrowhead indicates the position of the jB probe-induced shifted band; other nonspecific (ns) bands are indicated by arrows In the competition experiments (lanes 3–6 and 8–11), a 25- or a 250-fold molar excess of unlabeled wild-type (WT) or mutant (Mut) probe was used (B) Supershift assays in TM3 and TM4 cells using an anti-p50 (ap50) serum or an anti-p65 (ap65) serum The arrowhead and arrows are as in (A) above (C) ChIP assays of in vivo p65 and p50 binding to the jB site in nuclear extracts of testicular cells using antibodies against p65 (ap65) and p50 (ap50) A rabbit pre-immune serum (Pre) was included as a control.
Trang 8construct), and TM4 cells were used in the
co-transfec-tion experiments because TM4 cells had been shown
previously (Fig 8A) to be more responsive to TNF-a
induction No apparent effects of p50 overexpression
on luciferase activities were observed in the
F1R4-1-transfected cells (Fig 8B) The luciferase activity was
twofold higher than that of untransfected cells when
p65 was overexpressed; more significantly, the
lucifer-ase activities were further increlucifer-ased to threefold those
of untransfected cells when p50 and p65 were
co-over-expressed (Fig 8B) Consistent with the TNF-a
modulation demonstrated above (Fig 8A), TNF-a
treatment elevated the luciferase activities in
F1R4-1-transfected cells to a level comparable to that when
p65 was overexpressed, but lower than that when p50
and p65 were co-overexpressed (Fig 8B) On the other
hand, the luciferase activities were negligible and the
responsiveness to p50⁄ p65 NF-jB proteins and TNF-a
modulation was abolished when the F1MutkB jB
mutant was similarly assayed (Fig 8B), unequivocally
demonstrating positive modulation by NF-jB and TNF-a acting on the Rnf33 jB site
As Rnf33 is expressed in testicular cells, the effects
of TNF-a and p50⁄ p65 overexpression on Rnf33 tran-scription were directly assayed in these cells by real-time quantitative RT-PCR in TM4 cells Echoing the luciferase assay data above (Fig 8B), overexpression
of p50 resulted in an increase of only 40% in the Rnf33 transcript level, but p65, p50⁄ p65 co-expression
or TNF-a significantly upregulated Rnf33 transcription
by 2.4- to 2.6-fold in TM4 cells (Fig 8C), echoing the findings in luciferase assays However, the Rnf33 expression level did not change appreciably in the pres-ence of TNF-a in TM3 cells (Fig 8C), in agreement with the luciferase assay data in Fig 8A Taken together, data from luciferase assays and direct mea-surements of Rnf33 mRNA levels in TM3 and TM4 cells clearly demonstrate that TNF-a, p65 (probably in
a homodimeric form) or the p50–p65 NF-jB hetero-meric complex all serve to up-regulate Rnf33 expres-sion in testicular cells via the intronic jB motif located immediately downstream of exon 1 of Rnf33
Possible NF-jB regulated expression of Rnf33 in the pre-implantation embryo
In this study, Rnf33 transcriptional modulation was investigated in the testis and in two testicular cell lines The question remains whether NF-jB is also involved
in Rnf33 transcription in the oocyte and in the pre-implantation embryo where Rnf33 is expressed, as in the testis To investigate this possibility, the approxi-mate temporal expression profiles of the p65⁄ Rela and p50⁄ Nf-jb1 genes were examined based on bioinfor-matics analysis of the EST database in GenBank (Table 1) Mouse EST sequences for p65 are found in the oocyte, pre-implantation embryos and in the testis
On the other hand, p50⁄ Nf-jb1 EST sequences are found in the oocyte and in the testis but not in any of the pre-implantation embryos If NF-jB is experimen-tally shown in subsequent studies to be involved in transcriptional modulation of Rnf33 in oocyte and in early development as in the testis, it is likely that only the p65–p65 homodimer is involved, which is highly consistent with data presented in this work in the testis
Discussion
In a previous work [1,2], we have shown that in the fertilized egg and the zygote, Rnf33 transcription recruits three minor promoters (one of which is located upstream of the Rnf35 gene) and a major promoter,
0
2
4
6
8
10
12
14
16
RNF33
β-actin
p65
p105/p50
siRNA: NS p65 p50
NS p65 p50 SiRNA
1.00 0.52 0.85
1.00 0.98 0.68
1.00 0.71 0.98
A
B
Fig 7 Confirmation of NF-jB modulation of Rnf33 expression by
siRNA knockdown of p65 and p50 (A) p65 and p50 knockdown
and Rnf33 transcriptional down-regulation TM4 cells were
individu-ally transfected with a nonspecific (NS), p65 or p50 siRNA for 48 h
before real-time RT-PCR quantification of the relative mRNA levels;
the mRNA levels of the NS-treated TM4 cells were arbitrarily set
as 10 Data presented are from three independent experiments;
**P < 0.01 (B) RNF33 protein reduction on p65 knockdown siRNA
transfection was as described in (A) Representative western blots
of three independent experiments are shown The precursor p105
protein was used to represent p50 levels in the western blot
Dis-played below the blots are the computed relative levels of the
respective protein after normalization with the level of b-actin.
Trang 9designated P1 in Fig 1, which is dissected in this work
in the testis At the four- and eight-cell embryonic
stages, multiple promoter usage is resolved into the use
of only the major promoter, and this is followed by
complete Rnf33 gene silencing at the blastocyst stage
and the remaining phases of embryonic development
[2] Rnf33 is, however, reactivated specifically in the
testis in adult mice, as shown in this work We have
further shown that Inr sequences act as the core
promoter element for both the Rnf33 and Rnf35 genes
As Inr overlaps with the 5¢ end of exon 1, our studies
further attribute a critical role for noncoding
untrans-lated 5¢ exons and the acquired associated introns in
activating expression of intronless protein-encoding genes, as for retrogenes [24–26] Interestingly, mutating both the Inr element and the aTATA of Rnf33 led to the abolishment of only about 50% of promoter activi-ties in luciferase assays, strongly suggesting that the structure of the Rnf33 basal promoter is more complex than the discerned Inr and aTATA A 2-kb sequence that encompasses the upstream regulatory region, exon
1 and the solo intron of both Rnf35 and Rnf33 is found to be free from CpG islands (data not shown) The combined characteristics of the core promoters
of Rnf35 and Rnf33 are highly consistent with the general features of tightly regulated tissue-specific and
Table 1 Approximate expression profiles of p65 and p105 ⁄ p50 in the mouse pre-implantation embryos and the testis based on EST analy-sis Data shown are in transcripts per million.
p50 p65 TNF- α
– – – + – – – + – + + – – – +
RLU
F1R4-1 F1R4-1MutB TM4
* *
* *
* *
F1
F1R4-2/3
– TNF- α
*
Relative Rnf33 mRNA level
p50 p65 TNF- α
– – – + – – – + – + + –
– – + – – +
TM4 TM3
TM4
TM3
* *
* *
* *
RLU
A
Fig 8 jB-dependent TNF-a and p50⁄ p65 modulation of Rnf33 promoter activity (A) TNF-a up-regulation of Rnf33 promoter activity TM3 and TM4 cells were transfected with the jB-containing construct F1, with construct F1R4-2 ⁄ 3 from which jB had been deleted (see Fig 4A)
or with the jB mutant construct F1MutjB (see Fig 4B) TNF-a was added 24 h after transfection and the cells were cultured for a further 24-h period before being harvested for luciferase assays Open and hatched bars represent relative luciferase activities (RLU) in the absence
or presence of TNF-a, respectively (B) Transcriptional up-regulation by p50 and p65 overexpression TM4 cells were transfected with either construct F1R4-1 (see Fig 4A) or with the jB mutant construct F1R4-1MutjB, or were co-transfected with either the p50 or p65 overex-pression plasmid TNF-a was also included in the assay for comparison The cells were harvested for luciferase assays 48 h after transfec-tion or 24 h after treatment with TNF-a Open and gray bars represent luciferase activities of F1R4-1 and F1R4-1MutjB, respectively (C) TNF-a and p50⁄ p65 up-regulate Rnf33 transcription in testicular cells TM4 cells were transfected with p50 and ⁄ or p65 overexpression plasmids for 48 h before RNA was extracted and real-time quantitative RT-PCR assays for relative Rnf33 mRNA levels were carried out For analysis of the effect of TNF-a, both TM3 and TM4 cells were treated with TNF-a for 24 h before RNA was prepared and quantitative RT-PCR assays were carried out The Rnf33 mRNA level for the untreated TM4 cells was arbitrarily set as 1 Data presented are from three independent experiments and were analyzed using the Student’s t-test;*P < 0.05; **P < 0.01.
Trang 10temporal-specific promoters proposed based on
gen-ome-wide computation of the architecture of
mamma-lian promoters [27,28]
In this study, a jB element located in the only
intron of the Rnf33 gene was shown to be critical for
Rnf33 transcription; our data showed that the jB
ele-ment was targeted by the p65–p50 heterodimer and
possibly by the p65–p65 homodimeric complex, but
not by p50 alone, in the Sertoli cell-derived TM4 cells
(Fig 7B,C) NF-jB is a transcription factor inducible
by multiple stimuli to regulate a wide range of genes
Involvement of the NF-jB signaling pathway in the
regulation of genes involved in spermatogenesis and
other testicular functions in both Sertoli and Leydig
cells has been abundantly reported [16–20] Among the
five known NF-jB proteins, p50 and p65 are the major
NF-jB proteins expressed in the testis [21–23]
NF-jB-regulated expression of the testis-specific Rnf33 gene
echoes previous reports that expression of the
cAMP-response element-binding protein (CREB) and
andro-gen receptor (AR) andro-genes in Sertoli cells is regulated by
the NF-jB p65–p50 heterodimer or by p65 alone, but
not by p50 alone [16,17,29] Signaling pathways that
activate NF-jB have been well documented [13–15] In
the canonical NF-jB activation pathway, degradation
of IjBa through phosphorylation by the activated IjB
kinase (IKK) complex leads to the release of
cytoplas-mic NF-jB and nuclear relocation of NF-jB In an
IKK-independent pathway, external stimuli, including
hypoxia and genotoxic stresses, lead to NF-jB nuclear
localization Involvement of NF-jB in Rnf33
transcrip-tional modulation is consistent with the fact that testis
is a highly dynamic site of active and continuous
sper-matogenesis and is therefore under constant molecular
and evolutionary stresses Likewise, pre-implantation
development is also highly stressful However, the
NF-jB signaling stimuli and potential co-activator(s)
involved in the demonstrated NF-jB modulation of
Rnf33promoter activity in the testis and in early
devel-opment will need to be further identified
The consensus jB sequence is 5¢-GGGRHTYYCC-3¢
(in which R is purine, Y is pyrimidine and H is A, C
or T) A ‘phosphorylation code’ has been proposed for
p65 that targets NF-jB activity to specific subsets of
genes via the recognition of distinct groups of the
con-sensus jB site [30] In this code, the palindromic
5¢-GGGAATTCCC-3¢ jB sequence of Rnf33 was shown
to tolerate a wider range of differential
phosphoryla-tion of the amino-terminal Rel homology domain in
p65, hence providing the jB palindrome with a wider
choice of utilization of differentially phosphorylated
versions of p65 We also showed, in the luciferase
assay, that in TM4 cells there was a basal level of
promoter activity, and TM4 cells treated with TNF-a boasted the promoter activity (Fig 7C) as a result of NF-jB nuclear relocalization On the other hand, luciferase assays in both CHO-K1 and TM4 cells showed that despite the mutation in the jB site,30%
of the promoter activity remained (Figs 4B and 7B), indicating participation of other cis-acting element(s) and transcription factors in Rnf33 expression One can-didate cis element would be the adjacent HRE site tar-geted by HIF-1a Indeed, HIF-1a is transcriptionally regulated by NF-jB, thus establishing cross-talk between these two important transcription factors in the testis [12]
Studies have established that TNF-a is a major cyto-kine produced and released by germ cells and that TNF-a receptors are found on Sertoli and Leydig cells
of the testis [31] In the testis, TNF-a regulates sper-matogenesis [32], modulates Leydig cell steroidogenesis [33,34] and influences the expression of cell–cell adhe-sion molecules in Sertoli cells [35,36] There are abun-dant examples of involvement of the TNF-a⁄ NF-jB network in transcriptional modulation TNF-a induces NF-jB binding to the promoter of the AR gene and elevates AR promoter activities in Sertoli cells [17,29]
In Leydig cells, TNF-a-induced p50 and p65 specifi-cally interact with the CCAAT⁄ enhancer binding protein beta (C⁄ EBPb) to regulate the expression of Nur77, a regulator of steroidogenic-enzyme genes [18,20] In investigating activation of the
lipocalin-2gene, which is abundantly expressed in spermatogo-nial cells but expressed at only very low levels in Sertoli cells, Fujino et al [19] demonstrated regulation
of Sertoli cells by spermatogonial cell-mediated lipoca-lin-2 gene activation via an IKK-independent NF-jB pathway Expression of the Mullerian inhibiting sub-stance (MIS), a key molecule in sex differentiation and reproduction, is regulated by steroidogenic factor 1 (SF-1) also via the TNF-a⁄ NF-jB pathway [37] Most importantly, NF-jB up-regulates Fas expression in Sertoli cells, leading to apoptosis, a key event in the delicate balance of pro-apoptotic and anti-apoptotic signaling, to ensure optimal spermatogenesis [23,38] The finding that Rnf33 is also under NF-jB regula-tion in the testis is not surprising but funcregula-tionally rational Spermatogenesis is tightly regulated by a complex network of signals and stimuli and, as dis-cussed above, one of the important identified stimuli is NF-jB which, in turn, is also highly responsive to a wide range of external signals Furthermore, we have shown that the putative RNF33 protein interacts with the kinesin motor proteins KIF3A and KIF3B, possibly contributing to cargo mobilization along the microtubule [Huang, Huang, Chang, Hsu, Lin &