Nfil3 18030

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Gene ID: 18030
Gene symbol: nfil3
Gene Description: nuclear factor, interleukin 3, regulated
Aliases: AV225605; E4BP4
Human Ortholog Gene ID: [4783]
Reviewer Judgement: TF Gene
Functional Classification: DNA-Binding: sequence-specific
DBD Protein Group Classification: Zipper-Type Group
DBD Protein Group/Family Classification: Zipper-Type Group / Leucine Zipper Family
Homolog Cluster(s): 107503

Evidence:

Pubmed ID Function Species Evidence Strength Reviewer's Comments Reviewer
[7565758] DNA Binding; Transactivation Human Medium To define the transcriptional regulatory activity of NF-IL3A on the IL-3 promoter, transient transfections into MLA144 cells were performed. Introduction of the NF-IL3A BC12 expression plasmid into resting MLA144 T cells increased the CAT activity generated by the -315 and -173 plasmids 5.2- and 5.0-fold, respectively, compared with that generated by the BC12 expression vector alone. No significant change in the promoter activity of the -61 plasmid, which lacks the A region sequences, was seen. Introduction of 3-bp substitutions into the NF-IL3A consensus binding sequence eliminated responsiveness to NF-IL3A. Similarly, cotransfection of the NF-IL3A BC12 plasmid had no effect on the enhancerless pTE2 plasmid, but it activated the pTE2 plasmid containing two copies of the A region sequences approximately fivefold. These experiments demonstrate that the GTC nucleotides in the NF-IL3A consensus binding sequence (ATTACGTCTG) in the A region are required for responsiveness to NFIL3A. Specific binding of NF-IL3A to IL-3 promoter sequences was confirmed by competitive EMSA and DNase footprinting. Shannan HoSui
[10074173] DNA Binding; Transactivation Human Medium The binding specificity of E4BP4 was examined by competitive EMSA using a recombinant E4BP4 protein obtained by fusion of the E4BP4 open reading frame with that of the 1.3S subunit of the Proprionibacterium shermanii transcarboxylase. The results indicate that the sequence from nt 1650 to 1662 in box alpha was the binding site for E4BP4. This segment contains the sequence cTTACaTAAg (the lowercase letters represent the sequence which is different from the consensus E4BP4 binding sequence), which resembled the consensus E4BP4 binding sequence (A/G)T(G/T)A(T/C)GTAA(T/C) (12).

The binding specificity of overexpressed E4BP4 protein was examined in HuH-7 cells in transient-transfection assays. Total cell lysates from untransfected and transfected cells were obtained for Western and gel shift experiments. Two forms of E4BP4 were detected with anti-E4BP4 antiserum in pf:E4BP4-transfected cells. Box alpha binding activity was present in both untransfected cells and cells transfected with pf:E4BP4 or empty vector. In the nuclear extracts obtained from pf:E4BP4 transfectants, a supershift of box alpha binding activity by anti-FLAG and anti-E4BP4 antibodies was observed. This supershift was not seen with a control preimmune serum. These results indicated that E4BP4 could indeed bind to the box alpha sequence. They also showed that E4BP4 is not a major component of endogenous box alpha binding proteins. Moreover, inclusion of unlabeled competing oligonucleotides in molar excess showed that wild-type box alpha and the EF sequences could effectively abolish the box alpha binding activity that was supershifted with anti-FLAG antibody. Identical results were obtained with another E4BP4-transfected differentiated human hepatoma cell line, HepG2.

A single copy of the box alpha sequence in an upstream position stimulates the transcription of the HBV basal core promoter in both HepG2 and HuH-7 cells (68). To examine the effect of E4BP4 on box alpha , a CAT reporter plasmid containing an upstream box alpha sequence was cotransfected with pf:E4BP4 or pFLAG-CMV2 vector in HepG2 and HuH-7 cells. Coexpression of E4BP4 reduced the CAT activity by 35- and 14-fold in HepG2 and HuH-7 cells, respectively. Cotransfection with pFLAG-CMV2, which had no insert, had no significant effect.

Shannan HoSui
Additional Reviewer Comments:
Reviewer: Shannan HoSui

E4BP4, a bZIP transcription factor, has been shown to have both repressor and enhancer activity using reporter gene assays. Direct binding to a ATTACGTCTG consensus sequence was shown using EMSA and DNase footprinting.


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Facts about Nfil3 18030RDF feed
Gene description nuclear factor, interleukin 3, regulated  +
Gene id 18,030  +
Gene judgment TF Gene  +
Gene symbol nfil3  +, AV225605  +, and E4BP4  +
Gene taxonomy DNA-Binding: sequence-specific  +
Homolog cluster 107,503  +
Protein family 3 Zipper-Type Group/21 Leucine Zipper Family  +
Protein group 3 Zipper-Type Group   +
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