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

Inhibition of Interferon Signaling by the New York 99 Strain and Kunjin Subtype of West Nile Virus Involves Blockage of STAT1 and STAT2 Activation by Nonstructural Proteins

The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-α/β) and gamma IFN (IFN-γ) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-α. IFN-α signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins responsible for this inhibition

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

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

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

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