PMC

The diagram shows the UbC promoter region spanning from nt −371 (with respect to the transcription start site) to nt +876 cloned in the P3 reporter construct, including the proximal promoter (PP), the first exon and the unique intron of the 5′-UTR of the gene. The promoter fragment devoid of the intron (nt −371/+64), cloned into the P7 vector, is also highlighted.

(A) Schematic representation of the tested DNA constructs. UbC promoter elements contained in the reference construct P3 are the proximal promoter (PP, black solid line, nt −371/−1), the first noncoding exon 1 (Ex1, hatched box, nt +1/+64), and the 5′-UTR intron (Int, open line, nt +65/+876). –Int, construct devoid of the intron sequence. Int(s)-PP-Ex1 and Int(as)-PP-Ex1, constructs where the UbC intron sequence was moved upstream of the proximal promoter, either in the sense or antisense orientation. [Ex1-Int](s)-PP and [Ex1-Int](as)-PP, constructs where the exon 1-intron cassette was moved upstream of PP, in both sense and antisense orientation. (B) Constructs displayed in A were transiently transfected in HeLa cells and luciferase activities were measured 48 h afterwards, normalized on total protein content, and referred to the P3 construct, set equal to 100%. The graph data are the means (±SE) of five independent experiments. Statistical analysis revealed a highly significant difference (***, p<0.001) between P3 and all other constructs investigated.

(A) The schematic diagram shows the UbC intron region (nt +65/+876). ODNs and the relative positions of the putative transcription factor binding motifs are illustrated below: Sp1 binding sites are represented by open ovals and identified with a single-letter code, from (a) to (d); YY1 binding sites are represented by filled rectangles and named (e) and (f). Sequences of the different Sp1 and YY1 binding sites are shown and nucleotide substitutions introduced by mutagenesis are highlighted. (B) HeLa transient transfections with wild-type (P3) and the different Sp1 mutant luciferase constructs were carried out and luciferase expression evaluated by RT and quantitative RealTime-PCR at 48 h post-transfection as detailed under “Materials and Methods”. Promoter activity of the wild-type P3 was set to 100% and promoter activity of the mutants was expressed as a percentage of the wild-type construct. (C), (D) HeLa transient transfections with wild-type (P3) and the different YY1 mutant luciferase constructs were carried out and assayed as in B. Data presented are the means (±SE) of at least four different experiments, with two independent plasmid preparations. Asterisks indicate statistical differences (*, p<0.05; **, p<0.01; ***, p<0.001 versus P3). The statistically significant difference between YY1mut e–f and YY1mut e (C) is also indicated (*, p<0.05).

(A, Left) Schematic representation of full-length ODN 1 and of the three overlapping duplexes referred to as ODN 1a, ODN 1b and ODN 1c (28-, 28- and 27-bp long, respectively). Numbers in brackets refer to the position with respect to the major transcriptional start site, identified as +1. (Right) EMSA was performed with ³²P-labeled ODN 1 as the probe. HeLa nuclear extract (5 µg) was preincubated in the absence (–) or presence of a fifty-fold excess of wild-type or mutant competitors, before addition of the labeled probe, as indicated. (B, Left) Full-length ODN 5 and the two overlapping duplexes, 25-bp long, referred to as ODN 5a and ODN 5b. (Right) EMSA was performed as reported in A, with ³²P-labeled ODN 5 probe, in the absence (–) or presence of a fifty-fold excess of wild-type or mutant competitors, as indicated. (C, Left) Schematic representation of full-length and partial intron probe IV and of the four overlapping duplexes designed on the partial region and used for direct binding in gel shift experiments (ODN IVa, ODN IVb, ODN IVc and ODN IVd). ODN 4 used in previous studies was reported as well . (Right) Direct binding of HeLa nuclear extract (5 µg) with the ³²P-labeled ODNs (lanes 1–4) and demonstration of binding specificity to ODN IVb by using ODN IVb mut as the probe (lanes 5–6). (D, Left) Schematic representation of full-length and partial intron probe II and of the three overlapping duplexes designed on the partial region (ODN IIa, ODN IIb and ODN IIc), used in competitive EMSA experiments. (Right) EMSA was performed with ³²P-labeled ODN 3 as the probe, in the absence (–) or presence of a fifty-fold excess of wild-type or mutant competitors, as indicated. Arrows point to the Sp1 (A, B and C) or YY1 (D) bound probe. Representative EMSA are shown. Experiments were repeated three times with similar results.

(A) Efficiency of siRNA-mediated knockdown of YY1. HeLa cells were transfected with control nonsilencing siRNA oligo or with a YY1-specific siRNA. Quantitative RealTime reverse transcription PCR assays were done 48 and 72 h post-transfection. Expression data, normalized to B2M, were analyzed by the 2^−ΔΔC _T method and referred to the control, set equal to 1. Results shown in the graph are the means (±SE) of ten independent experiments. Asterisks indicate statistical significance versus control siRNA transfected cells, at each time point (***, p<0.001). (B) Western immunoblot of proteins from HeLa cells transfected with control or YY1-specific siRNA, at 48 and 72 h post-transfection. Equal amounts of total cellular proteins (20 µg) were loaded and immunoblotted for YY1. Blot was reprobed with α-tubulin as a loading control. The image is representative of three different experiments, with similar results. (C) Effects of YY1 depletion on expression of endogenous UbC gene. HeLa cells transfected with control or YY1-specific siRNA were analyzed by quantitative reverse transcription PCR, at 48 and 72 h post-transfection, for ubiquitin C RNA level. Expression data, normalized to B2M, are relative to the value of the control siRNA sample, set equal to 1. The histogram shows the means (±SE) of five different experiments. As in (A), asterisks indicate statistical significance versus control siRNA transfected cells, at each time point (*, p<0.05; **, p<0.01). (D) Effects of YY1 depletion on the UbC promoter-directed luciferase expression in HeLa cells. Cells cotransfected with P3, YY1mut e–f, or P7 reporter construct and control or YY1-specific siRNA, as labeled, were harvested at 72 h post-siRNA delivery and luciferase RNA level measured by Quantitative RealTime reverse transcription PCR. Expression data are relative to the value of P3 reporter vector in the control siRNA sample, set equal to 1. The graph shows the means (±SE) of at least ten independent experiments. Asterisks indicate statistical significance (***, p<0.001).

(A) Immunoblotting of proteins from HeLa cells transfected with YY1 expression vector (lanes 2, 4, 6) or control empty vector (lanes 1, 3, 5), at 48 h post-transfection. Nuclear (Nuc, 10 µg), cytosol (Cyt, 20 µg) and total (20 µg) extracts were obtained as reported under “Materials and Methods”. Arrows mark the YY1 and actin bands (upper and lower panel, respectively). Molecular weight standards (kDa) are indicated on the left. Actin was employed as the endogenous internal control. A representative blot is shown. Experiments were repeated three times with similar results. (B) EMSA performed with ³²P-labeled ODN IIa, containing a YY1 binding sequence, as the probe and HeLa nuclear extracts of cells transfected with control (-, lane 1) or YY1 expression vector (+, lane 2). The parentheses indicate the major nucleoprotein complexes. A representative image of three different EMSA is shown. Quantification of DNA-protein complexes was performed in a Molecular Imager and results are reported in the histogram as mean counts (±SE) of three different experiments (***, p<0.001). (C) Quantitative RealTime reverse transcription PCR analysis of endogenous ubiquitin C and luciferase mRNA levels in cells receiving YY1 expression plasmid (+YY1) or control empty vector (vector), performed at 48 h post-transfection. All the values are the means (±SE) of five different experiments.

(A) Schematic representation of the tested P3-SSmut construct. P3-SSmut is identical to the reference construct P3, except that the sequences of the 5′ and 3′ splice sites are mutated. The wild-type and mutant splice sites are displayed below the intron boundaries and the introduced nucleotide substitutions are highlighted in gray. The primers, which bridge the intron sequence, used for assessment of splicing by RT-PCR are indicated on the illustration: the forward primer was derived from the first exon of the 5′-UTR of UbC gene (black filled arrow) and the reverse primer is complementary to the luciferase (LUC) coding region (open arrow). (B) Gel image showing the results of RT-PCR analysis. The templates were cDNAs derived from P3 (wt, lane 3) or P3-SSmut (SSmut, lane 4) transfected cells, except for samples loaded in lane 1 and lane 2, where plasmids P3 and P7 were amplified in parallel to provide the size of the PCR products expected from unspliced (1416 bp) or spliced (651 bp) transcripts, respectively. M, DNA molecular weight marker (lane 5). The boxes separated by a line (on the left) indicate the expected size of unspliced transcripts, while the two adjacent boxes indicate the size of correctly spliced transcripts. The arrowhead highlights aberrantly spliced non specific products. The analysis is qualitative, but not quantitative, that is band intensity does not accurately reflect the relative abundance of the different transcripts. (C) Relative luciferase activity and (D) quantitative RealTime RT-PCR analysis of luciferase mRNA level from HeLa cells transfected with P3-SSmut or the wild-type construct P3. Data shown in the histograms are the means (±SE) of five independent experiments. Statistical analysis (t-test) revealed a highly significant difference (***, p<0.001) between P3- and P3-SSmut-directed luciferase expression.

(A) EMSA was performed with the ³²P-labeled ODN IIa as the probe, in the absence (–) or presence of a fifty-fold excess of wild-type or mutant competitors, as indicated. To confirm binding specificity, 1 µg of YY1 or Egr1 antibody was preincubated with nuclear extracts, before addition of the radiolabeled probe. Arrow points to the YY1 bound probe and the asterisk indicates the supershifted complex; ns marks a non specific nucleoprotein complex. (B) ChIP analysis of association of YY1 nuclear protein with the UbC intron region was performed using chromatin from formaldehyde-crosslinked HeLa cells immunoprecipitated with YY1 and Egr1 specific antibodies. ChIPed DNAs were analyzed by quantitative RealTime PCR with primers detecting the intron probe II region (+137/+336) containing the most proximal YY1 site, the downstream intron region (+608/+766) and the upstream promoter region (−781/−636). Data in the graph represent the means (±SE) of three independent immunoprecipitations, analyzed in triplicate. Asterisks indicate the statistical difference, calculated using t-test, between YY1 and Egr1 ChIPed samples (***, p<0.001). Representative gel for each primer set is shown above the histogram. (C) For Western-ChIP analysis, 20 µl of ChIP samples after de-crosslinking (lanes 2 and 3) were separated onto a 8% (w/v) polyacrylamide gel in parallel with a whole HeLa cell extract (lane 1) as a positive control, and probed with the anti-YY1 antibody. The gel photograph is representative of three repeated experiments, with similar outputs. The arrows on the right point to the YY1 and IgG immunoreactive bands. Molecular weight standards are indicated on the left. (D) Plasmid ChIPs were performed on HeLa cells transfected with P3Δ(−371/−38) plasmid, bearing either wild-type or mutant YY1 sites in the intron region. After 48 h ChIPs were performed with 5 µg of YY1 specific antibody or with no antibody added as a negative control to provide baseline values. After ChIP, the −37/+336 region of the human UbC promoter was amplified using RealTime qPCR with a plasmid backbone specific primer (RVprimer3 forward 5′-CTAGCAAAATAGGCTGTCCC-3′) and a primer complementary to a portion of the UbC promoter/intron sequence (intron probe II reverse 5′-CGCATTAGCGAAGGCCTCAAG-3′). PCR signals, representing YY1 occupancy, are normalized to the input and plotted as fold enrichment over the no Ab control. Results shown are the means (±SE) of three separate experiments, assayed in triplicate. The asterisks represent significant difference of wild-type versus mutant P3Δ construct (**, p<0.01).

(A) Absolute quantitative RealTime reverse transcription PCR for detection of unspliced luciferase transcripts upon mutagenesis of YY1 intronic binding sequences. HeLa cells transfected with the construct YY1mut e–f (carrying mutations in both YY1 binding sites) and with the wild-type P3 were harvested 48 h post-transfection and subjected to total RNA extraction, cDNA synthesis with random hexamers, and absolute quantification assay with two different primer pairs: LUC Fwd and LUC Rev, complementary to internal sites of luciferase coding sequence, were used to quantify total luciferase RNA copies (spliced and unspliced); intron probe VI-Fwd and LUC-1-Rev, which annealed within the intron sequence and the LUC coding region, respectively, were selected to measure only the unspliced luciferase transcripts. The graph shows the ratio of unspliced versus total luciferase RNA copies for both YY1 double mutant and wild-type reference construct, which was set equal to 1. The data are the means (±SE) of eight different experiments. Asterisk indicates statistical significance (t-test; *, p<0.05). (B) Effect of YY1 silencing on the splicing efficiency of the UbC intron. The absolute quantification assay described in A was performed on cDNAs obtained from HeLa cells cotransfected with P3 or YY1mut e–f reporter vector and YY1-specific or nonsilencing control siRNA, as indicated. Analysis was performed at 72 h post-siRNA transfection and results are expressed relative to the value obtained for the control siRNA sample, set as 1. The graph displays the means (±SE) of five different experiments. Asterisk indicates statistical significance (t-test; *, p<0.05). (C) Gel image showing the results of quantitative PCR displayed in B (lanes 1–4). P3 and P7 derived amplicons (lanes 5, 6) served as a reference for the unspliced or spliced transcripts, respectively. M, DNA molecular weight marker (lane 7) (D) Effect of splicing impairment on luciferase RNA decay. HeLa cells transfected with P3 or YY1mut e–f reporter construct were treated, 48 h post-transfection, with 5 µM actinomycin D. At the time points indicated, total RNA was extracted and analyzed by RealTime RT-PCR with the luciferase primer pair referred to above. Data, normalized to B2M, are expressed as a percentage of the time zero value detected for P3 or YY1mut e–f, respectively. P3, filled diamonds; YY1mut e–f, open squares. The graph shows the results of a typical RNA decay analysis. Similar results were obtained in three separate experiments. (E) Analysis of YY1 binding to UbC RNA by RIP, in HeLa cells. Immunoprecipitation with YY1 antibody or IgG (performed as described under Materials and Methods) was followed by qRT-PCR for UbC or the control RNA (18S rRNA). Upper panel, Etidium Bromide-stained gel. RT-PCR samples were loaded, as indicated. Lower panel, RT-PCR quantification of the indicated UbC fragments (exon 1 and 3′-UTR) and the 18S rRNA as a control. Data are shown as percent input and are the average±SE of six independent experiments (***, p<0.001).