SKP2 loss destabilizes EZH2 by promoting TRAF6-mediated ubiquitination to suppress prostate cancer.

EZH2 is crucial for the progression of prostate cancer (PCa) and castration-resistant prostate cancer (CRPC) through upregulation and activation of progenitor genes, as well as androgen receptor (AR)-target genes. However, the mechanisms by which EZH2 is regulated in PCa and CRPC remain elusive. Here we report that EZH2 is post-transcriptionally regulated by SKP2 in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of Skp2, Ezh2 and histone H3 lysine 27 trimethylation (H3K27me3) in both Pten null mouse embryonic fibroblasts (MEFs) and Pten null mouse prostate tissues. Loss of Skp2 resulted in a striking decrease of Ezh2 levels in Pten/Trp53 double-null MEFs and in prostate tumors of Pten/Trp53 double-null mutant mice. SKP2 knockdown decreased EZH2 levels in human PCa cells through upregulation of TRAF6-mediated and lysine(K) 63-linked ubiquitination of EZH2 for degradation. Ectopic expression of TRAF6 promoted the K63-linked ubiquitination of EZH2 to decrease EZH2 and H3K27me3 levels in PCa cells. In contrast, TRAF6 knockdown resulted in a reduced EZH2 ubiquitination with an increase of EZH2 and H3K27me3 levels in PCa cells. Furthermore, the catalytically dead mutant TRAF6 C70A abolished the TRAF6-mediated polyubiquitination of recombinant human EZH2 in vitro. Most importantly, a concurrent elevation of Skp2 and Ezh2 was found in CRPC tumors of Pten/Trp53 mutant mice, and expression levels of SKP2 and EZH2 were positively correlated in human PCa specimens. Taken together, our findings revealed a novel mechanism on EZH2 ubiquitination and an important signaling network of SKP2-TRAF6-EZH2/H3K27me3, and targeting SKP2-EZH2 pathway may be a promising therapeutic strategy for CRPC treatment.

Localization of regulatory protein binding sites in the proximal region of human myometrial connexin 43 gene.

Parturition is preceded by a large increase in gap junctions between myometrial smooth muscle cells. Connexin 43 is the major structural protein of myometrial gap junctions. To explore transcriptional regulation of the myometrial Cx43 gene, we used DNase I footprinting, electrophoretic mobility shift and transient transfection assays to examine a 312 bp promoter region (-164 to +148) of the gene, utilizing human myometrial cell cultures and nuclear extracts. The DNase I studies showed four regions of nucleoprotein interactions. Protection of region 1 (-80 to -31) encompassed an Activator Protein 1 (AP1) (-44 to -36) and two Specificity Protein 1 (Sp1) (-77 to -69 and -59 to -48) consensus sequences. Regions 2 to 4 included the transcription initiation site (-10 to +25), an Ets/NF-kB consensus sequence (+47 to +74) and a TA-rich region (+81 to +101) respectively. Gel mobility shift and supershift assays demonstrated c-Jun and Sp1 binding at the AP1 and Sp1 sites respectively. Promoter mutagenesis and transient transfection analyses combined with Sp1 and c-Jun/c-Fos over-expression studies indicate that both Sp1 and c-Jun are required for maximal promoter activity and, therefore, may positively regulate transcription of myometrial Cx43 during the initiation of labour.

Identification of a GABP alpha/beta binding site involved in the induction of oxytocin receptor gene expression in human breast cells, potentiation by c-Fos/c-Jun.

Oxytocin (OT) receptors (OTRs) mediate reproductive functions, including the initiation of labor and milk ejection. OTR messenger RNA levels are highly regulated, reaching the greatest concentration in the uterus at the end of gestation, and in the mammary gland during lactation. Factors directly effecting changes in OTR gene expression in the mammary gland are not known, so the present studies were done to elucidate possible regulators by characterizing the human OTR gene promoter and 5'-flanking sequence. By analyzing expression of promoter-luciferase constructs, we localized a region between -85 and -65 that was required for both basal and serum-induced expression in a mammary tumor cell line (Hs578T) that expresses inducible, endogenous OTRs. This DNA region contains an ets family target sequence (5'-GGA-3'), and a CRE/AP-1-like motif. The specific Ets factor binding to the OTR promoter was identified, by electrophoretic mobility immunoshift assays, to be GABP alpha/beta. Co-transfection of a -85 OTR/luciferase construct with vectors expressing GABP alpha and GABP beta1 had only a modest effect on expression, but cotransfection with GABP alpha/beta- with c-Fos/c-Jun-expressing plasmids resulted in an increase of almost 10-fold in luciferase activity. Mutation of either the GABP- or CRE-like binding sites obliterated the induction. These findings are consistent with the involvement of protein kinase C activity in serum induction of the endogenous gene in Hs578T cells. We showed the requirement for GABP alpha/beta and c-Fos/c-Jun in endogenous OTR gene expression, using oligonucleotide GABP and AP-1 binding decoys to inhibit serum-induced increases in 125I-labeled OT antagonist binding to Hs578T cells. Our work is the first characterization of the proximal promoter region of the human OTR gene, and it sets the stage for studying regulation of OTR expression in breast cells.

Transcriptional properties of RNA polymerase II within triplet repeat-containing DNA from the human myotonic dystrophy and fragile X loci.

Expansion of a (CTG)n segment within the 3'-untranslated region of the myotonic dystrophy protein kinase gene alters mRNA production. The inherent ability of RNA polymerase II to transcribe (CTG)17-255 tracts corresponding to DNA from normal, unstable, and affected individuals, and the normal (CGG)54 fragile X repeat tract, was analyzed using a synchronized in vitro transcription system. Core RNA polymerase II transcribed all repeat units irrespective of repeat length or orientation. However, approximately 50% of polymerases transiently halted transcription (with a half-life of approximately 10 +/- 1 s) within the first and second CTG repeat unit and a more transient barrier to elongation was observed roughly centered within repeats 6-9. Transcription within the remainder of the CTG tracts and within the CCG, CGG, and CAG tracts appeared uniform with average transcription rates of 170, 250, 300, and 410 nucleotides/min, respectively. These differences correlated with changes in the sequence-specific transient pausing pattern within the CNG repeat tracts; individual incorporation rates were slower after incorporation of pyrimidine residues. Unexpectedly, approximately 4% of the run-off transcripts were, depending on the repeat sequence, either 15 or 18 nucleotides longer than expected. However, these products were not produced by transcriptional slippage within the repeat tract.

Template end-to-end transposition by RNA polymerase II.

On 5'-template strand protruding templates, promoter-initiated run-off transcription by RNA polymerase II generates discrete, 15-16-nucleotide (nt) longer than expected products whose production is abrogated by elongation factor SII (Parsons, M. A., Sinden, R. R., and Izban, M. G. (1998) J. Biol. Chem. 273, 26998-27008). We demonstrate that template terminal complexes produce these RNAs and that transcript extension is a general and salt-sensitive (250 mM) feature of run-off transcription. On 5'-overhung templates the extended run-off transcripts appear to be retained within an RNA-DNA-enzyme ternary complex, and SII facilitates resumption of transcript elongation via a dinucleotide truncation intermediate. Moreover, on one of the 5-overhung templates, the initially extended complexes spontaneously resumed transcript extension and were uniquely resistant to salt (250 mM) challenge. However, SII did not facilitate this long distance extension on all template ends. Run-off transcripts on a blunt-ended template were initially extended by 2-11 nt (roughly in 2-nt increments); SII addition either before or after extension resulted in the accumulation of a 4-5-nt extension product. Based on these findings, we propose that the initial and continuously extended RNAs reflect intermediates and successful completion of template end-to-end transposition (template switching) by RNA polymerase II, respectively. Both the template end sequence and structure influenced the success of such an event.

RNA polymerase II ternary complexes may become arrested after transcribing to within 10 bases of the end of linear templates.

In the presence of elongation factor SII, arrested RNA polymerase II ternary complexes cleave 7-17 nucleotides from the 3'-ends of their nascent RNAs. It has been shown that transcription of linear templates generates apparent run-off RNAs, which are nevertheless truncated upon incubation with SII. By using high resolution gels, we demonstrate that transcription of blunt or 3'-overhung templates with RNA polymerase II generates two populations of ternary complexes. The first class pauses 5-10 bases prior to the end of the template strand. These complexes respond to SII by cleaving approximately 9-17 nucleotide RNAs from their 3'-ends and therefore may be termed arrested. A second class of complexes, which fail to respond to SII, transcribe to within 3 bases of the end of the template strand. These complexes appear to have run off the template since they have released their nascent RNAs. Run-off transcription occurs on all types of templates, but it is the predominant reaction on DNAs with 5'-overhung ends. Thus, RNA polymerase II ternary complexes that retain 5-10 bases of contact with the template strand down-stream of the catalytic site become arrested. Further reduction of downstream template contacts can lead to termination. We also show that the addition of Sarkosyl to the elongation reactions significantly changes the pattern of transcriptional arrest near the end of linear templates.

The active site of RNA polymerase II participates in transcript cleavage within arrested ternary complexes.

RNA polymerase II may become arrested during transcript elongation, in which case the ternary complex remains intact but further RNA synthesis is blocked. To relieve arrest, the nascent transcript must be cleaved from the 3' end. RNAs of 7-17 nt are liberated and transcription continues from the newly exposed 3' end. Factor SII increases elongation efficiency by strongly stimulating the transcript cleavage reaction. We show here that arrest relief can also occur by the addition of pyrophosphate. This generates the same set of cleavage products as factor SII, but the fragments produced with pyrophosphate have 5'-triphosphate termini. Thus, the active site of RNA polymerase II, in the presence of pyrophosphate, appears to be capable of cleaving phosphodiester linkages as far as 17 nt upstream of the original site of polymerization, leaving the ternary complex intact and transcriptionally active.

SII-facilitated transcript cleavage in RNA polymerase II complexes stalled early after initiation occurs in primarily dinucleotide increments.

RNA polymerase II ternary complex cleaves its nascent transcript in a 3'-->5' direction in the presence of elongation factor SII (Izban, M. G., and Luse, D. S. (1992) Genes & Dev. 6, 1342-1356; Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). We have characterized the cleavage products generated during the truncation process with a variety of stalled RNA polymerase II ternary complexes containing uniformly labeled transcripts. These complexes, which remain elongation competent, had stopped transcription because one nucleoside triphosphate was missing from the reaction mixture. Using a novel assay system, we demonstrate that cleavage occurs in predominantly dinucleotide increments, liberating 5'-phosphodinucleotides (pNpNs). In one instance with a particular C20 complex, the first cleavage event was equally partitioned between either a di-or trinucleotide increment with all subsequent truncations occurring by the preferred dinucleotide step. Our data indicate that both the kinetics and the exact increment of SII-facilitated transcript cleavage are influenced by transcript sequence.

The increment of SII-facilitated transcript cleavage varies dramatically between elongation competent and incompetent RNA polymerase II ternary complexes.

Elongation factor SII is required to increase the efficiency of transcription by RNA polymerase II through intrinsic arrest sites. RNA polymerase II ternary complexes exhibit a ribonuclease activity in the presence of SII, truncating nascent transcripts in a 3'-->5' direction. We show here that transcript cleavage is an obligatory step in re-establishing the elongation competency of complexes that have become blocked in elongation at an intrinsic arrest site. SII-facilitated transcript cleavage by these arrested complexes released 7-14 nucleotide RNA fragments. In contrast, SII-facilitated transcript cleavage by elongation competent complexes, which are stalled because of the absence of a nucleoside triphosphate from the reaction mixture, occurred primarily in dinucleotide increments. We can partially recreate the arrested phenotype and the preference for the large cleavage increment by stalling ternary complexes such that the 3'-end of the transcript contains consecutive U residues, which mimics the sequence of the 3'-ends of transcripts in arrested complexes.

Factor-stimulated RNA polymerase II transcribes at physiological elongation rates on naked DNA but very poorly on chromatin templates.

We used an in vitro assay system based on HeLa cell core transcription components to examine transcript elongation by RNA polymerase II on either naked DNA or chromatin templates as a function of the three known elongation factors, IIS, TFIIF, and TFIIX. We demonstrate for the first time that mammalian RNA polymerase II can achieve physiological elongation rates on naked DNA templates in vitro. The addition of TFIIF alone gave this rate, although IIS was required to minimize the block to elongation at intrinsic termination sites. However, IIS and TFIIF provided only a slight increase in the very poor elongation efficiency of RNA polymerase II on chromatin templates. The addition of TFIIX to reactions containing IIS and TFIIF reduced the elongation rate on naked DNA templates but slightly increased the elongation efficiency on chromatin. The ability of elongation factors either separately or in combination to stimulate transcription on naked DNA and chromatin templates was also examined.

The RNA polymerase II ternary complex cleaves the nascent transcript in a 3'----5' direction in the presence of elongation factor SII.

The process by which RNA polymerase II elongates RNA chains remains poorly understood. Elongation factor SII is known to be required to maximize readthrough at intrinsic termination sites in vitro. We found that SII has the additional and unanticipated property of facilitating transcript cleavage by the ternary complex. We first noticed that the addition of SII caused a shortening of transcripts generated by RNA polymerase II at intrinsic termination sites during transcription reactions in which a single NTP was limiting. Truncation of the nascent transcript was subsequently observed using a series of ternary complexes artificially paused after the synthesis of 15-, 18-, 20-, 21-, and 35-nucleotide transcripts. Transcripts as short as 9 or 10 nucleotides were generated in 5-min reactions. All of these shortened RNAs remained in active ternary complexes because they could be chased quantitatively. Continuation of the truncation reaction produced RNAs as short as 4 nucleotides; however, once cleavage had proceeded to within 8 or 9 bases of the 5' end, the resulting transcription complexes could not elongate the RNAs with NTP addition. Transcript cleavage requires a divalent cation, appears to proceed primarily in 2-nucleotide increments, and is inhibited by alpha-amanitin. The catalytic site of RNA polymerase II is repositioned after transcript cleavage such that polymerization resumes at the proper location on the template strand. The extent and kinetics of the transcript truncation reaction are affected by both the position at which RNA polymerase is halted and the sequence of the transcript.

Transcription on nucleosomal templates by RNA polymerase II in vitro: inhibition of elongation with enhancement of sequence-specific pausing.

The process by which RNA polymerase II elongates RNA chains in vivo, where the template is at least partially in a nucleosomal configuration, remains poorly understood. To approach this question we have partially purified RNA polymerase II transcription complexes paused early in elongation. These complexes were then used as substrates for chromatin reconstitution. Elongation of the nascent RNA chains on these nucleosomal templates is severely inhibited relative to elongation on naked DNA templates. Elongation on the nucleosomal templates results in a reproducible template-specific pattern of transcripts generated by RNA polymerase pausing. The RNA polymerases are not terminated because the large majority will resume elongation upon the addition of Sarkosyl or 400 mM KCl. The effectiveness of RNA polymerase II pause/termination sites is enhanced by the presence of nucleosomes. For example, a pause site similar in sequence to the c-myc gene exon 1 terminator is used four to seven times more effectively in reconstituted templates. A comparison of elongation on templates bearing phased nucleosomes and on reconstituted templates that show no predominant phasing pattern indicates that the locations of pause sites are not related to the positions of the nucleosomes. Rather, the major determinant of RNA polymerase pausing on the nucleosomal templates appears to be the underlying DNA sequence.

Derepression of a mouse alpha-fetoprotein expression vector in COS-1 cells by amplification of specific cis-acting sequences of the AFP promoter.

The existence of trans-acting regulatory factors has been demonstrated by in vivo competition with cis-acting sequences from both viral and eukaryotic genomes. Plasmids containing a functional SV40 origin of replication when transfected into permissive SV40 T-antigen producing COS-1 cells will amplify to high copy numbers (5,000 to 10,000) without inflicting toxic effects upon the host cell. This amplification vector (pSVori) has been used to amplify cis-acting regulatory elements which can act as competitors for positive and negative trans-acting factors in vivo. Using this amplification system we conducted experiments to determine whether amplification of alpha-fetoprotein (AFP) and albumin cis-acting promoter sequences could activate a corresponding co-transfected AFP-promoter-CAT or Alb-promoter-CAT expression vector in COS-1 cells. We used pMoMLV(-1009)AFPcat, or p(-308)Albcat-MoMLV as reporter genes and pSVori to amplify specific promoter sequences of the AFP or albumin promoter. Our experiments indicated that amplification of a region from -53 to -202 of the AFP promoter resulted in the activation of the pMoMLV(-1009)AFPcat and p(-308)Albcat-MoMLV expression vectors in COS-1 cells. Surprisingly, amplification of the albumin promoter sequences failed to activate either the pMoMLV(-1009)AFPcat or p(-308)Albcat-MoMLV plasmids.

Cell-specific expression of mouse albumin promoter. Evidence for cell-specific DNA elements within the proximal promoter region and cis-acting DNA elements upstream of -160.

Regulation of albumin gene expression is believed to be mediated by multiple nuclear factors that interact with cis-acting DNA sequences within the first 160 base pairs (bp) of the promoter. The minimal promoter sequence required to generate tissue-specific expression has not been clearly defined. We have constructed a series of transient expression vectors containing progressive deletions of the mouse albumin gene 5'-flanking sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and include the Moloney murine leukemia viral (Mo-MuLV) enhancer. Promoter activity was determined in mouse hepatoma and fibroblast cell lines by chloramphenicol acetyltransferase and S1 nuclease analyses. All constructions were compared with -623 Albcat-Mo-MuLV which contains all the sequence homology between the rat and mouse promoters. Low levels of expression were observed with -60 Albcat-Mo-MuLV (10%) in hepatoma but not fibroblast cells. Addition of promoter sequence to -208 bp progressively increased activity to 190% in the hepatoma cells, while -308 and -1612 Albcat-Mo-MuLV had activity similar to the -623 Albcat-Mo-MuLV level, and -3000 Albcat-Mo-MuLV showed a 2-fold reduction in transcriptional activity. The inclusion of promoter sequences upstream of -60 generated low levels of expression in the fibroblasts. We also show that factors from mouse liver nuclear extracts protect at least five regions of the albumin promoter upstream of -160. Our results indicate that tissue specificity is established within the proximal promoter region and that additional cis-acting elements that may have a functional role in the efficiency of albumin gene expression are located upstream of -160 bp.