Different Domains of the Transcription Factor ELF3 Are Required in a Promoter-specific Manner and Multiple Domains Control Its Binding to DNA

Janel L Kopp,Phillip J Wilder,Michelle Desler,Leo Kinarsky,Angie Rizzino

doi:10.1074/jbc.m609907200

Janel L Kopp, Phillip J Wilder + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m609907200

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Abstract

Elf3 is an epithelially restricted member of the ETS transcription factor family, which is involved in a wide range of normal cellular processes. Elf3 is also aberrantly expressed in several cancers, including breast cancer. To better understand the molecular mechanisms by which Elf3 regulates these processes, we created a large series of Elf3 mutant proteins with specific domains deleted or targeted by point mutations. The modified forms of Elf3 were used to analyze the contribution of each domain to DNA binding and the activation of gene expression. Our work demonstrates that three regions of Elf3, in addition to its DNA binding domain (ETS domain), influence Elf3 binding to DNA, including the transactivation domain that behaves as an autoinhibitory domain. Interestingly, disruption of the transactivation domain relieves the autoinhibition of Elf3 and enhances Elf3 binding to DNA. On the basis of these studies, we suggest a model for autoinhibition of Elf3 involving intramolecular interactions. Importantly, this model is consistent with our finding that the N-terminal region of Elf3, which contains the transactivation domain, interacts with its C terminus, which contains the ETS domain. In parallel studies, we demonstrate that residues flanking the N- and C-terminal sides of the ETS domain of Elf3 are crucial for its binding to DNA. Our studies also show that an AT-hook domain, as well as the serine- and aspartic acid-rich domain but not the pointed domain, is necessary for Elf3 activation of promoter activity. Unexpectedly, we determined that one of the AT-hook domains is required in a promoter-specific manner.

Highlights

Past several years, interest in one of its family members, Elf3, has grown considerably
We examined the ability of Elf3⌬355–363 to occupy the T␤R-II promoter in vivo by Chromatin Immunoprecipitation (ChIP), and we found that its presence is greatly reduced when compared with wild-type Elf3 (Fig. 1E)
In contrast to the pointed domain (PNT) domain, removal of the serine- and aspartic acid-rich (SAR) domain did not affect the expression level of Elf3 (Fig. 4C), but it significantly decreased the ability of Elf3 to stimulate transcription, ϳ30 – 40% (p Ͻ 0.05) upon comparing multiple experiments (Fig. 4B). These findings argue that Elf3 does not require the PNT domain to stimulate the promoter of the T␤R-II gene, whereas the SAR domain exerts a subtle, but significant, effect on Elf3 transactivation

Summary

EXPERIMENTAL PROCEDURES

Materials—Dulbecco’s modified Eagle’s medium was obtained from Invitrogen. Fetal bovine serum was purchased from HyClone Laboratories (Logan, UT). Cell Culture—As described previously, Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum was used in the culture of each cell line, including F9 embryonal carcinoma (EC) [11, 52] and 293T [11]. Promoter/Reporter (P/R) Gene Constructs—The P/R constructs mT␤R-II Ϫ108/ϩ56(B/A), mT␤R-II Ϫ108/ϩ56(B/x), and CMV-␤-gal have been described previously [11, 52, 53]. Expression Plasmids—Plasmid expression vectors for FLAGElf, FLAG-Elf3⌬N233, FLAG-Elf3⌬N270, FLAG-Elf3L142P, Ets, and the green fluorescent fusion (GFP) protein, GFP-Elf, have been described previously [12]. The plasmid expression vector for the Med23-(352– 625) fragment was obtained from

Primer pair used

RESULTS

Findings

DISCUSSION