The <i>vav</i>1 5′ untranscribed sequences contain cell-type specific cis-regulatory elements.

<p>(A) Expression of wild-type (wt) luciferase reporter gene (Le2) in cell lines from various tissue origins. Le2 was transfected into the cell lines as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029939#s2" target="_blank">Materials and Methods</a> and luciferase activity was measured 24 hr later. Data show luciferase activity normalized to <i>Renilla</i> transfection efficiency control and calculated relative to the luciferase activity of an empty vector expression, pGL3. The experiments were repeated five times. (B) Schematic map of the 5′ regulatory region of the human <i>vav</i>1 gene. Three putative transcription factor binding sites are highlighted by boxes. The changes introduced in these regions are as follows: nucleotide substitutions (red) and deletions (crooked lines). (C) The effect of these mutations/deletions was analyzed in Jurkat T cells, U937 myeloid cells and H441 lung cancer cells. Following transfection with plasmids containing luciferase under wt (Le2) or mutated <i>vav</i>1 promoter, the luciferase activity was measured and fold induction of activity was calculated relative to the activity of Le2. Experiments were repeated five times. Statistics were performed using the unpaired student T test. (**) indicates p<0.05 value and (***) indicates p<0.01.</p

Methylation of CpG sites in the <i>vav</i>1 promoter impairs expression of the reporter gene in various cell lines.

<p>(A) Le2 plasmid, either un-treated or methylated by CpG methyltransferase (M.SssI), was incubated with HpaII and analyzed on a gel. The plasmid treated with M.SssI was not digested by HpaII, indicating that methylation was successful. (B) Unmethylated or methylated Le2 was transfected into Jurkat T cells, U937 myeloid cells and H441 lung cancer cells. The luciferase activity of these plasmids was measured 24 hr after transfection. Fold induction of luciferase activity was calculated relative to the activity in cells transfected with unmethylated Le2. Each point is the mean of three experiments. (***) indicates p<0.01, unpaired student T test.</p

Methylation on CpG dinucleotides at putative transcription factor binding sites changes the affinity of protein complexes for the <i>vav</i>1 regulatory region.

<p>(A) EMSA was performed with Jurkat T cell nuclear extracts and lil3-4 labeled oligonucleotide. The probe was created by annealing complementary oligonucleotides lil79 and lil80 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029939#pone-0029939-t003" target="_blank">Table 3</a>).-3′). The following unlabeled competitors were added: unmethylated lil79-80 oligonucleotide (C₃C₄); oligo methylated on both CpG methylation sites (^metC₃^metC₄); oligo methylated only on CpG₃ (^metC₃C₄), or only on CpG₄ (C₃^metC₄). Competitor oligonucleotide was added in an amount equal to the labeled oligo (1∶1) or in 5 molar excess (1∶5).</p

Nucleotide sequence of the 5′ minimal regulatory region of the human <i>vav</i>1 gene.

<p>Boxes indicate putative binding sites for various transcription factors as predicted by bioinformatics. Their location is indicated relative to the transcription start site (+1 position). Putative sites for CpG methylation are highlighted in red, their arbitrary serial numbers are circled in green.</p

Mutations at the E2F/NF-e/c-Myb binding site affect binding of protein complexes to the <i>vav1</i> promoter <i>in vitro</i>.

<p>(A) Electrophoretic mobility shift assay (EMSA) with Jurkat nuclear extracts was performed in the presence of digoxigenin-labeled probe spanning nucleotides −45 to 0 of <i>vav1</i> promoter and containing E2F/NF-e/c-Myb and TCFα/PU.1/ELF1 binding sites (lil157-158; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029939#pone-0029939-t003" target="_blank">Table 3</a>). The competition assay was performed with the labeled oligonucleotide and unlabeled competitor oligonucleotides with point mutations as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029939#pone-0029939-t003" target="_blank">Table 3</a> in molar ratio of 1∶1 and 1∶5. The arrow shows the position of the complex that demonstrates sensitivity to the introduced mutations. (B) EMSA performed with labeled oligonucleotide containing only E2F/NF-e/c-Myb binding site (lil 87-88; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029939#pone-0029939-t003" target="_blank">Table 3</a>).</p

C-Myb is involved in regulation of <i>vav</i>1 expression in lung cancer cells.

<p>(A) Endogenous expression of <i>c-myb</i> mRNA in Jurkat T cells, H441 (<i>vav</i>1-positive) and H460 (<i>vav</i>1-negative) lung cancer cell lines was detected by RT-PCR and western blotting. (B) Empty vector pGL3 or the Le2 wt reporter construct was transfected either alone or with a c-Myb-expressing plasmid into H460 lung cancer cells (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029939#s2" target="_blank">Materials and Methods</a>). Luciferase activity was measured 24 hr after transfection (top panel). Luciferase activity is expressed as fold induction relative to basic pGL3 expression. Values are the mean of five independent experiments; significance was determined using the unpaired student T test. (***) indicates p<0.01. The bottom panel shows the level of <i>c-myb</i> and actin mRNA and protein expression in the transfected cells as determined by RT-PCR and Western blotting respectively. (C) H441 lung cancer cells were transfected with either scrambled DNA (-) or with siRNA against c-Myb. Seventy-two hours later, the mRNA levels of <i>c-myb</i>, <i>vav1</i> and <i>actin</i> were detected by RT-PCR.</p

Transfection conditions for different cell lines used in this study.

<p>Transfection conditions for different cell lines used in this study.</p

Primers that were used for gene expression analysis.

<p>Primers that were used for gene expression analysis.</p

Primers used for preparation of the <i>vav</i>1 promoter constructs^*.

<p>*The underlined sequences correspond to the nucleotide replacement mutations.</p

Mutations at various transcription factors binding sites affect protein complexes formation at the <i>vav1</i> promoter.

<p>Electrophoretic mobility shift assay (EMSA) with Jurkat and H441 nuclear extracts was performed in the presence of lil46-47 digoxigenin-labeled probe (nucleotides −98 to +28 of <i>vav1</i> promoter). To produce the mutant oligonucleotides, the corresponding mutated plasmids (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029939#pone-0029939-g002" target="_blank">Fig. 2B</a> schematic) were used as template for the PCR. A schematic of <i>vav</i>1 5′ regulatory sequences, exon 1 and relative oligonucleotide position is shown at the bottom. Bound protein complexes are numbered 1 to 5. The arrow shows the position of complex 5, the heaviest complex that is sensitive to the mutations introduced into the oligonucleotide sequence. The bottom panels of the figure schematically show the relative intensity of bands 1–5 of the EMSA experiment as determined by densitometry (ImageJ software).</p