Sequence-Specific Binding of Recombinant Zbed4 to DNA: Insights into Zbed4 Participation in Gene Transcription and Its Association with Other Proteins

Zbed4, a member of the BED subclass of Zinc-finger proteins, is expressed in cone photoreceptors and glial Müller cells of human retina whereas it is only present in Müller cells of mouse retina. To characterize structural and functional properties of Zbed4, enough amounts of purified protein were needed. Thus, recombinant Zbed4 was expressed in E. coli and its refolding conditions optimized for the production of homogenous and functionally active protein. Zbed4’s secondary structure, determined by circular dichroism spectroscopy, showed that this protein contains 32% α-helices, 18% β-sheets, 20% turns and 30% unordered structures. CASTing was used to identify the target sites of Zbed4 in DNA. The majority of the DNA fragments obtained contained poly-Gs and some of them had, in addition, the core signature of GC boxes; a few clones had only GC-boxes. With electrophoretic mobility shift assays we demonstrated that Zbed4 binds both not only to DNA and but also to RNA oligonucleotides with very high affinity, interacting with poly-G tracts that have a minimum of 5 Gs; its binding to and GC-box consensus sequences. However, the latter binding depends on the GC-box flanking nucleotides. We also found that Zbed4 interacts in Y79 retinoblastoma cells with nuclear and cytoplasmic proteins Scaffold Attachment Factor B1 (SAFB1), estrogen receptor alpha (ERα), and cellular myosin 9 (MYH9), as shown with immunoprecipitation and mass spectrometry studies as well as gel overlay assays. In addition, immunostaining corroborated the co-localization of Zbed4 with these proteins. Most importantly, in vitro experiments using constructs containing promoters of genes directing expression of the luciferase gene, showed that Zbed4 transactivates the transcription of those promoters with poly-G tracts

Sequence-specific binding of recombinant Zbed4 to DNA: insights into Zbed4 participation in gene transcription and its association with other proteins.

Zbed4, a member of the BED subclass of Zinc-finger proteins, is expressed in cone photoreceptors and glial Müller cells of human retina whereas it is only present in Müller cells of mouse retina. To characterize structural and functional properties of Zbed4, enough amounts of purified protein were needed. Thus, recombinant Zbed4 was expressed in E. coli and its refolding conditions optimized for the production of homogenous and functionally active protein. Zbed4's secondary structure, determined by circular dichroism spectroscopy, showed that this protein contains 32% α-helices, 18% β-sheets, 20% turns and 30% unordered structures. CASTing was used to identify the target sites of Zbed4 in DNA. The majority of the DNA fragments obtained contained poly-Gs and some of them had, in addition, the core signature of GC boxes; a few clones had only GC-boxes. With electrophoretic mobility shift assays we demonstrated that Zbed4 binds both not only to DNA and but also to RNA oligonucleotides with very high affinity, interacting with poly-G tracts that have a minimum of 5 Gs; its binding to and GC-box consensus sequences. However, the latter binding depends on the GC-box flanking nucleotides. We also found that Zbed4 interacts in Y79 retinoblastoma cells with nuclear and cytoplasmic proteins Scaffold Attachment Factor B1 (SAFB1), estrogen receptor alpha (ERα), and cellular myosin 9 (MYH9), as shown with immunoprecipitation and mass spectrometry studies as well as gel overlay assays. In addition, immunostaining corroborated the co-localization of Zbed4 with these proteins. Most importantly, in vitro experiments using constructs containing promoters of genes directing expression of the luciferase gene, showed that Zbed4 transactivates the transcription of those promoters with poly-G tracts

A. Structure modeling for BED zinc-finger (I, II, III and IV) domains of Zbed4, based on the structure of ZBED1 (PDB ID: 2ct5).

<p>The program iMol, version 0.40 and the UCSF Chimera package <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035317#pone.0035317-Pettersen1" target="_blank">[39]</a> were used to generate these models. <b>B</b>. Superimposed model of all predicted BED zinc-finger structures of Zbed4 (I, II, III and IV) and ZBED1 (PDB ID: 2ct5). Each finger is shown in a different color.</p

Determination of the minimal poly-G tract required for interaction with Zbed4 and of the affinity of Zbed4 for two different GC-box consensus sequences.

<p>Zbed4 (20 µg) was incubated with each of different 20-mer ssDNA primers containing G-tracts flanked by a different number of poly-As (0.5 nM), and with each of two GC-box consensus sequences for 45 min. The 20 µl reaction mixtures were loaded on a 1% agarose gel and EMSA was carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035317#pone-0035317-g005" target="_blank">Figure 5</a>. A single primer (0.5 nM) and Zbed4 (20 µg) were used as controls. 1 kb DNA ladder was used as a standard for nucleic acid size. A. Agarose gel stained with SYBR Gold. B. The same gel stained with Coomassie R-250. As seen, the affinity of Zbed4 binds to for the oligonucleotides increases with the increasing number of Gs in the tracks. that contain at least 5 Gs and Quantification of the Zbed4-oligonucleotide complex bands showed that those with 5–11 Gs, 12–16 Gs and 17–18 Gs have similar density values: 105,415±15,217; 167,810±13,671; and 214,727±15,861, respectively. The complex with 19Gs has the highest value: 360,835. In addition, Zbed4 binds only to the GC-box, GGGGCGGGGC, indicating that the neighboring nucleotides of the core sequence are critical.</p

Zbed4 interacts with SAFB1 <i>in vitro</i> and <i>in vivo</i>.

<p><b>A.</b> Membrane overlay assay: Proteins were resolved on SDS-PAGE and transferred to a PVDF membrane. Lane 1: cell lysate; lane 2: purified Zbed4 protein; lanes 3, 4 and 5: cell lysate incubated with Zbed4 protein (overlay). Lanes 1, 2 and 3 were probed with the Zbed4 antibody and lanes 4 and 5 with the SAFB1 and ERα antibodies, respectively. <b>B. </b><i>Subcellular co-localization of Zbed4 with SAFB1</i>, carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035317#s4" target="_blank">Materials and Methods</a>. The merged image clearly shows that Zbed4 co-localizes with SAFB1 in the nucleus of Y79 retinoblastoma cells. DAPI was used to stain the nuclei. <b>C.</b> Co-immunoprecipitation experiments were performed using Y79 retinoblastoma cell extracts. Both Zbed4 and SAFB1 were detected in the immunoprecipitated proteins obtained with Zbed4 antibody (left panel) or SAFB1 antibody (right panelIn each case, each duplicate lane of the blots obtained after SDS-PAGE of the immunoprecipitated proteins was incubated with Zbed4 or SAFB1 antibodies. Immunoprecipitation experiments were performed using Y79 retinoblastoma cell extracts (Input) and antibodies against Zbed4 or SAFB1. Duplicate aliquots of the proteins immunoprecipitated by each antibody were immunoblotted after SDS-PAGE with antibodies against Zbed4 (lanes 2 and 4) or SAFB1 (lanes 3 and 5). Both Zbed4 and SAFB1 are detected in each immunoprecipitate. Aliquots of Input material also show on the Western blots the presence of Zbed4 (lane 1) and SAFB1 (lane 6).</p

Co-localization of Zbed4 with ERα and MYH9 in Y79 retinoblastoma cells, carried out as described in Materials and Methods.

<p>The immunostaining results indicate that both ERα and MYH9 co-localize with Zbed4 in these cells. The major difference is that in A co-localization is seen in the nuclei and cytoplasm for Zbed4 and ERα whereas in B it is only observed in the cytoplasm for Zbed4 and MYH9. C. Immunoprecipitation experiments were carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035317#pone-0035317-g007" target="_blank">Figure 7C</a> with Y79 retinoblastoma cells (Input, lanes 1 and 6) but using antibodies against Zbed4 (lanes 1, 2 and 4) or MYH9 (lanes 3, 5 and 6) instead; both Zbed4 and MYH9 are detected in each immunoprecipitate.</p

Far-UV CD spectrum of Zbed4 (black curve) and curve obtained using the CONTINLL program (white curve).

<p>The CONTINLL-calculated curve conforms well to the experimental spectra of Zbed4. SELCON and CDSSTR-calculated curves (not shown) were essentially identical to that of CONTINLL.</p

Zbed4 mediates transcriptional activation of several genes expressed in retina.

<p><b>A.</b> Immunocytochemical detection of endogenous Zbed4 in HEK293 cells (upper image) and in HEK293 cells stably transfected with a Zbed4 expression construct (lower image). The endogenous Zbed4 in HEK293 cells is localized mainly in the nuclei (cyan) and barely detected in the cytoplasm whereas in the HEK293 stable-transfected cells Zbed4 is seen in both, the nuclei (cyan) and cytoplasm (green). DAPI was used to stain nuclei. <b>B</b>. Trans-activation of promoters from genes expressed in retina by Zbed4. Stably transfected HEK 293 cells expressing Zbed4 and non-transfected HEK 293 cells were used in transient transfections with luciferase reporter constructs carrying different retinal promoters. Luciferase activity was measured in the cell lysates and normalized for each transfection system to the corresponding β-galactosidase activity for each sample. The results are expressed as fold induction by Zbed4 of the mean luciferase activity of the uninduced promoter compared to that of the Vim/<i>luc</i>, Rho/<i>luc,</i> Blue/<i>luc</i>, Green/<i>luc</i>, and α-PDE/<i>luc</i> reporter constructs, respectively, ± S.D. p values for each pair of Zbed4 stimulated and non-stimulated promoters are noted above the bars.</p

Fractionation and purification of recombinant Zbed4 protein expressed in <i>E. coli</i> cells.

<p><b>A.</b> SDS-PAGE. 50 µg/well of total protein from each fraction obtained in the expression, purification and refolding of Zbed4 were separated by SDS-PAGE on 4–12% Bis-Tris gels and stained with Coomassie R-250. <i>Lane 1,</i> whole <i>E. coli</i> lysate before IPTG induction. <i>Lane 2,</i> whole cell lysate after 6 h induction by IPTG. <i>Lane 3,</i> soluble proteins of <i>E. coli</i> cell lysate in HEPES buffer containing 1% Triton X100 and other components (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035317#s4" target="_blank">Materials and Methods</a>), after passing through a French pressure chamber and centrifugation at 150,000 g. <i>Lane 4</i>, insoluble material (inclusion bodies) of lysate. <i>Lane 5</i>, solubilized inclusion bodies in 6 M Gu-HCl buffer 1, after centrifugation at 150,000 g. <i>Lane 6</i>, insoluble fraction of inclusion bodies. <i>Lane 7</i>, Zbed4 purified using BD Talon Co²⁺-activated affinity chromatography, after the refolding procedure and concentration. <i>Lane 8</i>, Novex Sharp (Invitrogen) standard protein markers. <b>B.</b> Detection of Zbed4 on Western blots using Penta His antibodies. Following SDS-PAGE, the separated proteins of each fraction were transferred to PVDF membranes and after blocking and incubation with Penta His antibodies conjugated with horseradish peroxidase, Zbed4 was visualized with the ECL Substrate of the Fast Western blot kit.</p

Relative affinity of recombinant Zbed4 for the different oligonucleotides.

<p>Zbed4 (20 µg) was incubated with 0.5 nM different 20-mer ssDNA (left panel) and ssRNA oligonucleotides (right panel) for 45 min and the whole reaction mixtures (20 µl) with the protein-DNA complexes were subjected to EMSA on 1% agarose gels run at room temperature in HEPES buffer, pH 8.2, at 20 mA. <b>A</b>. Agarose gel stained using SYBR Gold for detection of nucleic acid retardation. <b>B.</b> The same gel stained with Coomassie R-250 for detection of Zbed4 protein. Zbed4 (20 µg) and a single primer (0.5 nM) were applied separately to the gel as controls. 1 kb DNA ladder was used as a standard for nucleic acid size. As seen, Zbed4 binds only to DNA and RNA 20-mers that contain poly-Gs.</p