Eaf2 106389

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Gene ID: 106389
Gene symbol: eaf2
Gene Description: ell associated factor 2
Aliases: AW048865; Festa; FESTA-L; FESTA-S; Traits; U19
Human Ortholog Gene ID: [55840]
Reviewer Judgement: TF Gene Candidate
Functional Classification: Transcription Factor Binding: tf co-factor binding;

DNA-Binding: sequence-specific

Homolog Cluster(s): 106389

Evidence:

Pubmed ID Function Species Evidence Strength Reviewer's Comments Reviewer
[12446457] Transactivation; TF PPI Human Medium EAF2 interacts with ELL in vivo as demonstrated by coimmunoprecipation experiments in human 293 and HeLa cells. To determine the region of ELL that interacts with EAF2, the authors transfected a series of FLAG-tagged constructs that contained the amino-terminal, middle, and carboxy-terminal regions of ELL. Transfected 293 cells were immunoprecipitated with the FLAG antibody and probed with the EAF2 (1F11) monoclonal. Endogenous EAF2 coprecipitated with the amino-terminal region of ELL (amino acids 1-210), but not with the carboxy-terminus of ELL (amino acids 401-621). A series of deletion mutants were then used to map the interaction domain more precisely.

ELL and EAF2 colocalize in nuclear speckles in experiments using HeLa and 293 cell lines with EAF2 and ELL antibodies.

EAF2 contains a transactivation domain in its carboxy terminus. Fusions to the GAL4 DNA binding domain with full-length EAF2 and EAF2 deletion mutants were generated and cotransfected into 293 cells with either a GAL4-E1bCAT or a control E1bCAT reporter construct and a beta -galactosidase plasmid. GAL4-tkCAT or a control tkCAT promoter were also tested. Full-length EAF2 could function as a transactivator. Deletion mutants mapped this activity to amino acids 177-260 of EAF2. This carboxy-terminal region of EAF2 showed the strongest activity, exhibiting a 23.3 � 8.9-fold increase in activation compared with the control.

Shannan HoSui
[16114057] DNA Binding; Transactivation; TF PPI Human Medium Full-length U19/Eaf2 and various U19/Eaf2 deletion mutants were cloned into the PM vector, yielding expression vectors for fusion proteins of GAL4 DNA binding domain (DBD) with full-length U19/Eaf2 or U19/Eaf2 deletion mutants. Each construct was cotransfected with the luciferase reporter plasmid pER-luc and an

internal control Renilla luciferase expression vector into COS-7 cells. Full-length U19/Eaf2 transactivates more than sevenfold luciferase reporter gene expression compared to GAL4-DNA binding domain alone. Multiple regions of Eaf2 were capable of modulating transactivation.

To assess sequence specific DNA binding, GST�U19/Eaf2 fusion protein were generated and a random binding site selection (RBSS) experiment was performed. A 6bp sequence, ACTTTA, was recognized by GST�U19/Eaf2 fusion protein. Specificity of U19/Eaf2 binding was verified by competitive electrophoretic mobility shift assay (EMSA). Wild-type Tbox containing oligomers competed for the binding of GST-U19/Eaf2 to the labeled probe whereas the mutated T-box oligomers did not.

Interaction between U19 and ELL was confirmed by co-immunoprecipitation using COS-7 cells.

Shannan HoSui
Additional Reviewer Comments:
Reviewer: Shannan HoSui

Eaf2 interacts with the transcription factor ELL, as shown by co-immunoprecipitation and subcellular localization experiments. Multiple regions of Eaf2 have been shown to transactivate reporter gene constructs. DNA binding was verified using a random binding site selection experiment that identified a hexamer motif recognized by Eaf2, as well as by EMSA. However, transactivation via direct binding of Eaf2 to the hexamer has not been shown.


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Facts about Eaf2 106389RDF feed
Gene description ell associated factor 2  +
Gene id 106,389  +
Gene judgment TF Gene Candidate  +
Gene symbol eaf2  +, AW048865  +, Festa  +, FESTA-L  +, FESTA-S  +, Traits  +, and U19  +
Gene taxonomy Transcription Factor Binding: tf co-factor binding  +, and DNA-Binding: sequence-specific  +
Homolog cluster 106,389  +
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