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

Abstract

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.

The increasing diversity of functions attributed to the SAFB family of RNA-/DNA-binding proteins

“…Conversely, weaker interactions may be maintained in the presence of low salt, but there may be a risk of artefactual interactions. [10], hnRNPC [10], hnRNPD [120], hnRNPG [25,84,121], hnRNPI [10], hnRNPK [10,122], hnRNPU [10] SR proteins SRSF1 [33], SRSF7 [26], SRSF9 [26,33], SREK1 [88], SRRM1 [123] SR protein kinase SRPK1 [89] Chromatin CHD1 [72], NCOR [37,71], HDAC3, [37,71] BRG1 [73], Matrin3 [54] Transcription RNAPolII [33], TAF II 68 [124], steroid receptors [67][68][69], P53 [70] Miscellaneous PIAS1 [37], ZO-2 [125], Zbed4 [126] Also, co-immunoprecipitation does not distinguish between direct protein interactions and interactions mediated by 'bridging' molecules, such as other proteins or nucleic acids. Formation of homo-and hetero-dimers of SAFB1 and SAFB2, as well as interactions with several hnRNP proteins, has been observed (see Table 1 for a summary of some of the proteins shown to interact with SAFB proteins).…”

ZBED2 is an antagonist of interferon regulatory factor 1 and modifies cell identity in pancreatic cancer

“…The ZBED gene family encodes nine zinc finger-containing TFs in humans, which originated from a domesticated DNA transposase gene from an hAT transposable element (28). While lacking in transposase activity, human ZBED TFs instead retain their zinc finger domain to perform sequence-specific DNA binding and function as transcriptional regulators in a cell-type specific manner (29)(30)(31). Within this family, ZBED2 is one of the least-understood members, in part because of its recent evolution and lack of a mouse ortholog (28).…”

TLR4/NF-κB-Responsive MicroRNAs and Their Potential Target Genes: A Mouse Model of Skeletal Muscle Ischemia-Reperfusion Injury

“…Mouse Zbed4, like its human ortholog, has 2 nuclear receptor-interacting motifs (LXXLL) characteristic of coactivators/corepressors of nuclear hormone receptors [ 60 ]. Zbed4 has been reported to interact with estrogen receptor alpha (ER α ) and cellular myosin 9 (MYH9) in retinoblastoma cells [ 61 ]. However, association of Zbed4 with innate immunity has not yet been reported.…”