Identification of the DNA binding element of the human ZNF300 protein

Abstract

The human ZNF300 gene is a member of the KRAB/C2H2 zinc finger gene family, the members of which are known to be involved in various developmental and pathological processes. Here, we show that the ZNF300 gene encodes a 68-kDa nuclear protein that binds DNA in a sequence-specific manner. The ZNF300 DNA binding site, C(t/a)GGGGG(c/g)G, was defined via a random oligonucleotide selection assay, and the DNA binding site was further confirmed by electrophoretic mobility shift assays. A potential ZNF300 binding site was found in the promoter region of the human IL-2Rβ gene. The results of electrophoretic mobility shift assays indicated that ZNF300 bound to the ZNF300 binding site in the IL-2Rβ promoter in vitro. Transient co-transfection assays showed that ZNF300 could activate the IL-2Rβ promoter, and that the activation was abrogated by the mutation of residues in the ZNF300 binding site. Identifying the DNA binding site and characterizing the transcriptional regulation property of ZNF300 would provide critical insights into its potential as a transcriptional regulator.

Overexpression of the human ZNF300 gene enhances growth and metastasis of cancer cells through activating NF‐kB pathway

“…Transient transfection was performed as previously reported [17]. To test the transcriptional regulation of ZNF300 at the TRAF2 gene promoter, the expression vector pCDNA‐ZNF300 was co‐transfected into HeLa cells with pGL3‐TRAF2 or a luciferase reporter driven by a composite promoter containing three copies of the wild‐type NF‐κB responsive element (three times NF‐κB‐Luc) [34].…”

ZNF300, a recently identified human transcription factor, activates the human IL-2Rβ promoter through the overlapping ZNF300/EGR1 binding site

“…That study also showed that ZNF300 has the same binding affinity to the DNA sequences CTGGGGGCG and ACGGGGGCG [10]. Further EMSA was carried out to detect whether changing the first nucleotide (an A → G, C or T change) of ACGGGGGCG would affect the binding affinity of ZNF300 to this consensus.…”

“…Recombinant ZNF300 and Egr1 share binding sites in vitro In our previous study, we found that ZNF300 binds specifically to a GC-rich element (C(t/a)GGGGG(g/c)G) through its zinc finger domains [10]. That study also showed that ZNF300 has the same binding affinity to the DNA sequences CTGGGGGCG and ACGGGGGCG [10].…”

“…Expression and purification of the GST-ZF300 fusion protein, the GST-Egr1 fusion protein and GST The GST-ZF300 fusion protein was expressed and purified as previously reported [10]. The GST-Egr1 expression plasmids were transformed into E. coli BL21 (DE3) cells.…”

“…Thus, it is difficult to study the function of ZNF300 by genetic means using an animal model. Our recent studies have showed that ZNF300 recognizes the GC-rich DNA element (C(t/a)GGGGG(g/c)G), which is similar to the consensus binding site of Egr1 (GCG(G/T)GGGCG) [10]. Egr1, encoded by the immediate early growth response gene, is rapidly induced by growth factors to transduce the proliferative signal.…”