Nuclear factor kappa B and tumor necrosis factor-alpha modulation of transcription of the mouse testis- and pre-implantation development-specific Rnf33⁄Trim60 gene Kong-Bung Choo1,2,3, Min-Chuan Hsu1,2, Yao-Hui Tsai1,4, Wan-Yi Lin1 and Chiu-Jung Huang4 Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan Department of Biotechnology and Laboratory Science in Medicine, Taipei Veterans General Hospital, Taipei, Taiwan Institute of Clinical Medicine, National Yang Ming University, Taipei, Taiwan Department of Animal Science and Graduate Institute of Biotechnology, School of Agriculture, Chinese Culture University, Yangmingshan, Taipei, Taiwan 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 28613100 Tel: +886 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 composed of a short noncoding exon and an intronless coding exon 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 modulation, a 2.5-kb Rnf33 sequence, inclusive of the upstream regulatory region, exon and the associated intronic sequence, was dissected in transient transfection and luciferase assays An initiator and an atypical TATA-box were shown to act as the core promoter elements of the gene Deletion and mutagenesis of the 2.5-kb sequence in luciferase constructs further demonstrated that an intronic and palindromic kappa B (jB) sequence was an important cis element 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 factora was directly demonstrated in TM3 and TM4 cells by real-time PCR quantification 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] 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-implantation embryo of the mouse; both genes are silenced at the blastocyst stage before placental implantation and 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, hypoxia-inducible factor 1a; HRE, hypoxiaresponse 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 FEBS Journal 278 (2011) 837–850 ª 2011 Chinese Culture University Journal compilation ª 2011 FEBS 837 NF-jB modulates testis-specific Rnf33 expression K.-B Choo et al remain 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 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 promoter is TATA-less but utilizes an initiator (Inr) sequence as the core promoter element Two transcription 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 [3,4] We have also shown, in a previous 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 cotranscription 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 transcriptional regulation of the Rnf33 retrogene First of Rnf35 promoter NF-Y CDP Inr Rnf33 Rnf35 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 transcription 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 downregulation Our findings suggest possible functional involvement of the putative RNF33 protein in spermatogenesis 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-implantation-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 seminiferous 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 Sertoli cells of the testis kb Rnf33TSS’s Rnf35 P1 Rnf33 Fig 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 838 FEBS Journal 278 (2011) 837–850 ª 2011 Chinese Culture University Journal compilation ª 2011 FEBS K.-B Choo et al NF-jB modulates testis-specific Rnf33 expression Te T3M TM4 Li Rnf33 β-actin Fig Testicular expression of Rnf33 RNA samples were prepared from the mouse testis (Te) and from the testicular cell lines TM3 and TM4 for use in RT-PCR analysis with Rnf33-specific primers Liver (Li) was included as a negative control, and b-actin was used as a PCR control Identification 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 kb of upstream regulatory sequence, the 245-bp noncoding exon and 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 luciferase 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 luciferase 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 transcriptional activities, indicating the presence of other important transcriptional cis sequences in the neighborhood This was supported by the detection of only 10% transcriptional suppression following transfection 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 transcriptional regulatory elements in the retained exon and possibly also in the intron sequence This hypothesis gained support when progressive deletions of the intronic 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 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, indicating enhancer-like functions (Fig 3B, US-R4F and DS-R4F) Furthermore, the enhanced transcriptional activities were additive: up to three- or fivefold up-regulation 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 orientation, 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-acting transcriptional element(s) in the R4 sequence residing 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 Putative 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 factor 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 predicted TFBSs, the three R4 subsections were either retained or deleted individually or in different combinations from the F1 construct for luciferase assays FEBS Journal 278 (2011) 837–850 ª 2011 Chinese Culture University Journal compilation ª 2011 FEBS 839 NF-jB modulates testis-specific Rnf33 expression K.-B Choo et al A B Fig 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 contained the upstream regulation region, exon and kb of the Rnf33 intron; the sequences were cloned in front of the promoter-less luciferase 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 wildtype 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) orientation as the luciferase (Luc) gene, as displayed The constructs were individually transfected into CHO-K1 cells for luciferase assays The luciferase activity of the parental pGL3 promoter was arbitrarily set as 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 (Fig 4A, left-hand panel) Deletion of R4-2 or R4-3 alone (constructs F1R4-1 ⁄ 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 (construct F1R4-1) resulted in an increase, of 50%, in 840 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 FEBS Journal 278 (2011) 837–850 ª 2011 Chinese Culture University Journal compilation ª 2011 FEBS K.-B Choo et al NF-jB modulates testis-specific Rnf33 expression A Fig Association of R4 transcriptional activity with a jB element (A) Further mapping of transcriptional activities to a subsection (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 constructs displayed on the left, the R4 segment in F1 is magnified for clarity by omitting the sequence between exon and R4 (denoted by the slanting double-break symbols in the construct displays) (B) Identification of jB as the major cis transcriptional 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 downward-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 B C F1R4-3 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 transcriptional element(s) to the 93-bp R4-1 sequence To investigate the contribution of the three discerned putative TFBSs in R4-1 to Rnf33 transcriptional 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 transcription cannot be ruled out because HIF-1a is also a target gene of NF-jB [11,12] On the other hand, FEBS Journal 278 (2011) 837–850 ª 2011 Chinese Culture University Journal compilation ª 2011 FEBS 841 NF-jB modulates testis-specific Rnf33 expression mutating the putative GATA-binding site (construct F1MutG) had no discernible effects on transcriptional activity, ruling out a role of the putative GATA-binding 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) Promoter-activity analysis in luciferase assays firmly established 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 targeted 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 established 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 testicular 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 prepared from the TM3 and TM4 cells, a protein-induced band shift was observed (Fig 6A, lanes 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, and 9) A jB mutant oligonucleotide, however, had little effect on the observed band shift (Fig 6A, 842 K.-B Choo et al A Te TM3 TM4 Li Te TM3 TM4 Li 0.78 0.76 0.83 3.16 3.05 0.74 p50 p65 B p50 RL: p65 RL: β-actin Fig 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 Liver (Li) was included as a control in the analyses 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 protein-induced bands in both TM3 and TM4 cells were obliterated, indicating specific p65 targeting (Fig 6B, lanes 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 and 5) In in vivo p65-binding assays carried out by chromatin immunoprecipitation (ChIP), the anti-p65 and -p50 sera both yielded various 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 omitted, 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 transcriptional 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 transcriptional 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 FEBS Journal 278 (2011) 837–850 ª 2011 Chinese Culture University Journal compilation ª 2011 FEBS K.-B Choo et al NF-jB modulates testis-specific Rnf33 expression A TM4 TM3 NE (g): – + + + + + + + + + + Mut Competitor: – – WT Mut – WT B TM4 TM3 p50: – + – – + – C p65: – – + – – + t k pu oc p6 p5 e In M a- a- Pr Testis ns ns TM3 ns TM4 Liver 10 11 Fig 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 probeinduced 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 transfected 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 siRNA-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%, respectively, 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 nonspecific 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 siRNA did not result in appreciable reduction of RNF33 protein (Fig 7B, RNF33 panel) The results of the siRNA experiments clearly support p65-modulated Rnf33 expression and indicate that p50 seems to play a lesser role than p65 in Rnf33 expression Taken together, our data demonstrate that Rnf33 transcription is modulated by the NF-jB p65 protein, probably in the form of the more ubiquitous p65–p50 heterodimer, 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 ⁄ (from which the jBcontaining R4-1 segment had been deleted) or with the jB mutant construct F1MutkB (see Fig for constructs), 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 luciferase activity in F1-tranfected TM4 cells relative to the untreated cells (Fig 8A) Consistent with previous assays in CHO-K1 cells, luciferase activities were negligible 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 promoter activation in testicular cells is modulated by TNF-a and the modulation is dependent on the presence 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 for FEBS Journal 278 (2011) 837–850 ª 2011 Chinese Culture University Journal compilation ª 2011 FEBS 843 NF-jB modulates testis-specific Rnf33 expression A Relative mRNA level 16 14 12 10 NS p65 p50 SiRNA ** ** ** p65 p50 Rnf33 B siRNA: NS p65 1.00 p65 p50 0.52 0.85 1.00 0.98 0.68 1.00 0.71 0.98 p105/p50 RNF33 β-actin Fig 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 individually 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 Displayed below the blots are the computed relative levels of the respective protein after normalization with the level of b-actin construct), and TM4 cells were used in the co-transfection 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 F1R4-1transfected cells (Fig 8B) The luciferase activity was twofold higher than that of untransfected cells when p65 was overexpressed; more significantly, the luciferase activities were further increased to threefold those of untransfected cells when p50 and p65 were co-overexpressed (Fig 8B) Consistent with the TNF-a modulation demonstrated above (Fig 8A), TNF-a treatment elevated the luciferase activities in F1R4-1transfected 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 844 K.-B Choo et al 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 transcription were directly assayed in these cells by realtime 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 presence 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 measurements 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 heteromeric complex all serve to up-regulate Rnf33 expression in testicular cells via the intronic jB motif located immediately downstream of exon 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 preimplantation embryo where Rnf33 is expressed, as in the testis To investigate this possibility, the approximate temporal expression profiles of the p65 ⁄ Rela and p50 ⁄ Nf-jb1 genes were examined based on bioinformatics 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 experimentally 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, FEBS Journal 278 (2011) 837–850 ª 2011 Chinese Culture University Journal compilation ª 2011 FEBS K.-B Choo et al NF-jB modulates testis-specific Rnf33 expression A TM3 F1 F1R4-2/3 – TNF-α + TNF-α F1MutκB 0.5 RLU 1.5 B + – – + – TM4 F1R4-1 F1R4-1MutB – – RLU C p50 p65 TNF-α – – – + TM4 ** * + – – + RLU – + – + TM4 + – + – – ** – – ** * + – + ** ** p50 p65 TNF-α – – – – ** ** TM3 Relative Rnf33 mRNA level Fig 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 ⁄ 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 overexpression plasmid TNF-a was also included in the assay for comparison The cells were harvested for luciferase assays 48 h after transfection 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 RTPCR assays were carried out The Rnf33 mRNA level for the untreated TM4 cells was arbitrarily set as Data presented are from three independent experiments and were analyzed using the Student’s t-test;*P < 0.05; **P < 0.01 Table Approximate expression profiles of p65 and p105 ⁄ p50 in the mouse pre-implantation embryos and the testis based on EST analysis Data shown are in transcripts per million Gene UniGene no Oocyte Zygote Cleavage Morula Blastocyst Testis p65 p105 ⁄ p50 Mm.249966 Mm.256765 51 51 140 36 0 57 42 25 designated 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 untranslated 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 activities 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 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 FEBS Journal 278 (2011) 837–850 ª 2011 Chinese Culture University Journal compilation ª 2011 FEBS 845 NF-jB modulates testis-specific Rnf33 expression temporal-specific promoters proposed based on genome-wide computation of the architecture of mammalian 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 element 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-jBregulated expression of the testis-specific Rnf33 gene echoes previous reports that expression of the cAMPresponse element-binding protein (CREB) and androgen receptor (AR) 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 cytoplasmic 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 transcriptional modulation is consistent with the fact that testis is a highly dynamic site of active and continuous spermatogenesis 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 Rnf33 promoter activity in the testis and in early development 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 consensus jB site [30] In this code, the palindromic 5¢GGGAATTCCC-3¢ jB sequence of Rnf33 was shown to tolerate a wider range of differential phosphorylation 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 846 K.-B Choo et al 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 candidate cis element would be the adjacent HRE site targeted 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 cytokine 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 spermatogenesis [32], modulates Leydig cell steroidogenesis [33,34] and influences the expression of cell–cell adhesion molecules in Sertoli cells [35,36] There are abundant 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 specifically 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 lipocalin2 gene, which is abundantly expressed in spermatogonial cells but expressed at only very low levels in Sertoli cells, Fujino et al [19] demonstrated regulation of Sertoli cells by spermatogonial cell-mediated lipocalin-2 gene activation via an IKK-independent NF-jB pathway Expression of the Mullerian inhibiting substance (MIS), a key molecule in sex differentiation and reproduction, is regulated by steroidogenic factor (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 regulation in the testis is not surprising but functionally rational Spermatogenesis is tightly regulated by a complex network of signals and stimuli and, as discussed 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 & FEBS Journal 278 (2011) 837–850 ª 2011 Chinese Culture University Journal compilation ª 2011 FEBS K.-B Choo et al Choo, submitted] The microtubule transportation system, coupled with intercellular junctions, is essential in the translocation and positioning of spermatids in the spermatogenesis process [39,40] Kinesin proteins have indeed been shown to be present at junctions along adjacent microtubules of spermatids and Sertoli cells, thus contributing to spermatid translocation [41–43] Hence, demonstration of Rnf33 expression in Sertoli cells under the regulation of NF-jB may imply functional involvement of the RNF33 protein in spermatogenesis In summary, the present study demonstrates that Rnf33 expression in the testis is regulated by an intronic jB sequence modulated by the NF-jB subunits p65 and p50, and also by TNF-a Although p65 is probably expressed in the oocyte and the pre-implantation embryo, it remains to be shown if the TNF-a ⁄ NF-jB signaling pathway also contributes to transcriptional regulation of Rnf33 in pre-implantation development Experimental procedures NF-jB modulates testis-specific Rnf33 expression were quantified by real-time RT-PCR using the DyNAm Flash SYBRỊ Green qPCR kit (Finnzymes, Espoo, Finland) RNA samples extracted from treated cells were reverse transcribed as described above Real-time PCR was performed in a LightCyclerÒ 480 (Roche, Mannheim, Germany) in 96-well plates The reaction mixture was 20 lL of cDNA, · SYBR Green PCR Master Mix (Finnzymes) and 0.4 lm each of forward and reverse primers PCR primers for Rnf33 were F1466 and Rnf33-qPCR-R (5¢-GTTCTTAGAGGTCCA TAGGTGACA-3¢) For normalization, the mRNA level of the glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene in each RNA preparation was determined using primers GAPDH-F (5¢-GCCTCCTGCACCACCAACTG-3¢) and GAPDH-R (5¢-CCAGTAGAGGCAGGGATGATGT-3¢) The real-time PCR program was: pre-incubation at 50 °C for min; initial denaturation at 95 °C for min; and 45 cycles at 95 °C for 10 s, 63 °C for 15 s and 72 °C for 30 s The program was terminated by a final extension at 60 °C for and cooling at 40 °C for The relative Rnf33 mRNA levels were normalized to the mRNA level of the reference Gadph gene The melting curve of the amplification product was always checked to ensure a single clean peak that represented good-quality real-time PCR data Cell lines and mice The testicular TM3 and TM4 cell lines were obtained from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan; the cell lines were originally obtained by BCRC from the American Type Culture Collection (ATCC, Manassas, VA, USA) for maintenance and distribution in Taiwan The cells were cultured in a : mixture of Ham’s F12 medium and Dulbecco’s modified Eagle’s medium containing 1.2 gỈL)1 of sodium bicarbonate, 4.5 gỈL)1 of glucose (TM3 only) and 15 mm Hepes, and 5% (v ⁄ v) horse serum and 2.5% fetal bovine serum C3H mice were used in this work and were maintained at the Laboratory Animal Centre of the National Yang Ming University, Taipei This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Taipei Veterans General Hospital, and the mice were killed according to IACUC guidelines RT-PCR expression profiling and real-time quantitative RT-PCR To determine Rnf33 expression, RT-PCR was applied as previously described [1] After the oligo(dT)-primed reverse transcription reaction, PCR was carried out using primer F1466 (5¢-GTGTGTGTCAAGCCCACTTTTCTG-3¢) of the coding sequence and primer R1863 (5¢-GTGGG TGGTGGATTTTGTTGTTTG-3¢) of the 3¢-UTR sequence of Rnf33 to generate a 398-bp PCR product PCR was performed for 35 cycles using an annealing temperature of 65 °C and an extension time of 30 s for each cycle Mouse b-actin primers were used as a control Cellular Rnf33 mRNA levels Construction of luciferase reporter plasmids and site-specific mutagenesis The 2560-bp F1 genomic fragment was PCR-amplified from the mouse genomic BAC45 clone [2] using the DyNAzymeÔ II Hot Start DNA Polymerase (Finnzymes), and the PCR product was cloned into the promoter-less pGL3Basic luciferase vector at the NheI and XhoI restriction sites Short deletions and site-specific mutations were carried out using the commercial PhusionÔ Site-Directed Mutagenesis kit (Finnzymes), following the manufacturer’s instructions The mutations and deletions in the constructs were confirmed by sequencing Transient transfection, TNF-a treatment and luciferase assays Transient transfection was performed using the PLUSÔ Reagent and LipofectamineÔ (Invitrogen, Carlsbad, CA, USA), as previously described [3,4] Luciferase assays were performed 48 h post-transfection using the Dual-LuciferaseÒ Reporter 1000 Assay kit (Promega, Madison, WI, USA) in a 96-well microtiter plate, as instructed by the manufacturer The plate was read using a luminometer Transfection was typically carried out in duplicate, and for each transfection sample, the luciferase assay was also carried out in duplicate Three or more independent experiments of transfection and the associated luciferase assay were performed for each construct The p50 and p65 expression plasmids were a gift from Dr Neil Perkins; the genes were under transcriptional regulation of the long FEBS Journal 278 (2011) 837–850 ª 2011 Chinese Culture University Journal compilation ª 2011 FEBS 847 NF-jB modulates testis-specific Rnf33 expression terminal repeat (LTR) of Rous sarcoma virus (RSV) [44] In experiments in which TNF-a was used, TNF-a (SigmaAldrich, St Louis, MO, USA) was typically added to the untransfected cells or to the cells 24 h post-transfection, to a final concentration of 20 ngỈmL)1, and the cells were further incubated for 24 h before being harvested for luciferase assays or RT-PCR analysis The Student’s t-test was used for statistical analysis of the luciferase assay data; values of P < 0.05 were considered significant Preparation of nuclear extracts and total protein lysates Nuclear extracts and protein lysates were prepared essentially as previously described [3,4] TM3 and TM4 cells cultured in 100-mm dishes were harvested and the cell pellets were washed three times with NaCl ⁄ Pi before being gently suspended in 300 lL of cold buffer A (10 mm Hepes, pH 7.9, 10 mm KCl, 1.5 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm phenylmethanesulfonyl fluoride) The cell suspension was kept on ice for 15 followed by the addition of CA630 (Sigma-Aldrich) to a final concentration of 0.5% and then the cell suspension was briefly vortexed The samples were spun down at 10 000 g for 30 s at °C and the cell pellets were resuspended in 80 lL (cultured cells) or 120 lL (for the testis) of ice-cold buffer C (20 mm Hepes, pH 7.9, containing 0.42 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 25% glycerol, 0.5 mm dithiothreitol and 0.5 mm phenylmethanesulfonyl fluoride) The suspension was vigorously rocked at °C for 15 on a shaking platform before centrifugation at 10 000 g for 20 at °C Aliquots of the supernatant obtained were kept at )70 °C until used For total protein lysates, mouse testis or cultured cells were resuspended in 120 or 80 lL, respectively, of cold buffer C for 15 on a shaking platform Total protein lysates were cleared by centrifugation at 10 000 g for 20 at °C, aliquoted and kept at )70 °C until used EMSA and supershift assays The EMSA probes were prepared by end-labeling singlestranded oligonucleotides with [32P]dATP[cP] using T4 DNA kinase (Promega) followed by annealing the complementary strands of the oligonucleotides at 20 pmolỈlL)1, as previously described [45] The binding reaction mixture contained 100 mm Tris, 500 mm KCl, 10 mm dithiothreitol, 2.5% glycerol, mm MgCl2, 50 ngỈlL)1 of poly(dIỈdC), 0.05% Nonidet P-40, 40 fmol of 32P-labeled probe and lg of nuclear extracts, and the binding reaction was allowed to proceed at room temperature for 30 In competition assays, a 25- or a 250-fold molar excess of unlabeled double-strand oligonucleotide was added to the binding reaction and the reaction was also allowed to proceed at room temperature for 30 Oligonucleotides containing the jB 848 K.-B Choo et al sequence (underlined) that were used in the competition experiments were: wild type, 5¢-AGGTCTGGGAATTCCC CCCGGA-3¢; and mutant 5¢-AGGTCTGGGAATagggCCC GGA-3¢ (mutated nucleotides shown in lowercase letters) For supershift assays, 2.5 lg of a polyclonal anti-p65 serum (sc-109x) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or an anti-p50 serum (sc-7178x) (Santa Cruz), was added to the reaction mixture and the reaction was incubated at room temperature for 20 In all cases, binding complexes were displayed on 6% polyacrylamide gels, followed by blotting onto a positively charged nylon membrane Probe signals on the membrane were detected using a Typhoon 8000 Molecular Dynamics PhosphoImager (Amersham Pharmacia Biotech, Bucks., UK) ChIP ChIP was performed essentially as previously described [3] After incubation with specific antibodies, the immunocomplexes were incubated at 68 °C in a water bath overnight to reverse the cross-links in the samples, followed by digestion with 10 lg of each of RNase A and proteinase K at 42 °C for h After digestion, DNA samples were purified by phenol ⁄ chloroform extraction followed by ethanol precipitation The DNA pellet was dissolved in 10 lL of Tris–EDTA buffer Three-microliter aliquots of each DNA sample were used in PCR analysis in the presence of 40 pmol each of the Rnf33-specific RNF33-ChIP-F (5¢-AG GGCATAAAGGAGGGCAGGGAAC-3¢) and RNF33ChIP-R (5¢-CATCAGCTTCCCTTATGAGAACAG-3¢) primers in 10-lL PCR reaction volumes The PCR was performed for 33 cycles at an annealing temperature of 65.6 °C to generate a 300-bp amplification product that was shown in a 1.5% agarose gel Transfection with siRNA To knock down p50 or p65 expression, p50 (sc-29408), p65 (sc-29411) or a nonspecific negative-control (sc-37007) double-stranded RNA (Santa Cruz) was transfected individually into · 105 TM4 cells in a 3.5-cm petri dish using Lipofectamine 2000 (Invitrogen), according to the manufacturers’ instructions Briefly, cells were first washed twice with serum-free (SF) medium before transfection The siRNA oligonucleotides and lL of Lipofectamine 2000 were prepared separately in 250 lL of SF medium The siRNA oligonucleotide was then added slowly into Lipofectamine 2000 and the mixture was incubated for 20 at room temperature before being added to the cells in the presence of 500 lL of SF medium A final siRNA oligonucleotide concentration of 80 nm was routinely used Six hours post-transfection, the SF medium was replaced with complete medium After 48 h of transfection, cells were harvested for total RNA and protein lysate preparations FEBS Journal 278 (2011) 837–850 ª 2011 Chinese Culture University Journal compilation ª 2011 FEBS K.-B Choo et al Acknowledgements We thank Dr Neil D Perkins, University of Bristol, Bristol, UK, for the p50 and p65 expression plasmids This work was supported by the National Science Council (Taiwan) grants NSC95-2311-B-075-001 and NSC96-2311-B-075-001 to K.B.C and C.J.H as coprincipal investigators References Choo KB, Chen HH, Liu TY & Chang CP (2002) Different modes of regulation of transcription and pre-mRNA processing of the structurally juxtaposed homologs, Rnf33 and Rnf35, in eggs and in preimplantation embryos Nucleic Acids 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RelA and p50 ⁄ NFjB1 nuclear factor- jB (NF-jB) subunits, and the jB-dependent transcription was also modulated by tumor necrosis factor alpha (TNF-a) p65 ⁄ p50 transcriptional modulation of Rnf33... in both the Leydig and Sertoli cells of the testis kb Rnf33TSS’s Rnf35 P1 Rnf33 Fig Map of the Rnf35 and Rnf33 genes The relative map positions of the intronless genes (boxes) are as established... targeted 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 ⁄