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

Overexpression of Human γC-crystallin 5 bp Duplication Disrupts Lens Morphology in Transgenic Mice

Expression of c.119_123dup CRYGC, associated with autosomal dominant human cataracts, causes degeneration and vacuolization of lens fiber cells and cataracts in transgenic mice. This confirms the causative nature of this mutation and suggests that it acts through a direct toxic effect on lens fiber cells

Direct Transcriptional Effects of Apolipoprotein E

A major unanswered question in biology and medicine is the mechanism by which the product of the apolipoprotein E ε4 allele, the lipid-binding protein apolipoprotein E4 (ApoE4), plays a pivotal role in processes as disparate as Alzheimer's disease (AD; in which it is the single most important genetic risk factor), atherosclerotic cardiovascular disease, Lewy body dementia, hominid evolution, and inflammation. Using a combination of neural cell lines, skin fibroblasts from AD patients, and ApoE targeted replacement mouse brains, we show in the present report that ApoE4 undergoes nuclear translocation, binds double-stranded DNA with high affinity (low nanomolar), and functions as a transcription factor. Using chromatin immunoprecipitation and high-throughput DNA sequencing, our results indicate that the ApoE4 DNA binding sites include ∼1700 gene promoter regions. The genes associated with these promoters provide new insight into the mechanism by which AD risk is conferred by ApoE4, because they include genes associated with trophic support, programmed cell death, microtubule disassembly, synaptic function, aging, and insulin resistance, all processes that have been implicated in AD pathogenesis. SIGNIFICANCE STATEMENT This study shows for the first time that apolipoprotein E4 binds DNA with high affinity and that its binding sites include 1700 promoter regions that include genes associated with neurotrophins, programmed cell death, synaptic function, sirtuins and aging, and insulin resistance, all processes that have been implicated in Alzheimer's disease pathogenesis

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

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

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

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