Archives of Biochemistry and Biophysics 569 (2015) 54–61

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Characterization of the transcriptional activation domains of human TEF3-1 (transcription enhancer factor 3 isoform 1) Cheng Qiao 1, Yajie Jiang 1, Cuilan Deng, Zebo Huang, Kaixuan Teng, Lan Chen, Xin Liu ⇑ Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China

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Article history: Received 8 October 2014 and in revised form 23 January 2015 Available online 14 February 2015 Keywords: Transcription enhancer factor 3 isoform 1 Transcriptional activation domains Yeast HUVEC Angiogenesis

a b s t r a c t TEF3-1 (transcription enhancer factor 3 isoform 1) is a human transcriptional factor, which has a N-terminal TEA/ATTS domain supposedly for DNA binding and C-terminal PRD and STY domains for transcriptional activation. Taking advantage of the efficient reporter design of yeast two-hybrid system, we characterized the TEF3-1 domains in activating gene expression. Previously study usually mentioned that the C-terminal domain of TEF3-1 has the transcriptional activity, however, our data shows that the peptides TEF3-11–66 and TEF3-1197–434 functioned as two independent activation domains, suggesting that Nterminal domain of TEF3-1 also has transcriptional activation capacity. Additionally, more deletions of amino acids 197–434 showed that only the peptides TEF3-1197–265 contained the minimum sequences for the C-terminal transcriptional activation domain. The protein structure is predicted to contain a helix-turn-helix structure in TEF3-11–66 and four b sheets in TEF3-1197–265. Finally, after the truncated fragments of TEF3-1 were expressed in HUVEC cells, the whole TEF3-1 and the two activation domains could increase F-actin stress fiber, cell proliferation, migration and targeted gene expression. Further analysis and characterization of the activation domains in TEF3-1 may broaden our understanding of the gene involved in angiogenesis and other pathological processes. Ó 2015 Elsevier Inc. All rights reserved.

Introduction TEF3-12, which is also named related transcriptional enhancer factor 1 (RTEF-1) and TEAD4 [1,2], is a member of the transcription enhancer factor 1 family whose members have a commonly conserved TEA/ATTS domain for DNA binding. There are four family members in the TEAD family, namely TEAD1, TEAD2, TEAD3 and TEAD4 [3–5]. These proteins also share a common ‘‘CAAT’’ (MCAT) binding site [6] but function differently in different tissues and different biological programs through unclear molecular mechanism. Human TEF3-1 (TEAD4, Genbank number NM_003213) is expressed mostly in cardiac and skeletal muscle and has the potential to induce hypertrophy and to regulate cardiac and skeletal gene expression through the MCAT elements [2,7,8]. It has been recently reported

⇑ Corresponding author at: School of Pharmaceutical Sciences, Wuhan University, 185 East Lake Road, Wuhan 430071, China. Fax: +86 27 68759850. E-mail address: [email protected] (X. Liu). 1 The two authors contribute equally to the paper. 2 Abbreviations used: TEF3-1, transcription enhancer factor 3 isoform 1; TEAD4, TEA domain family member 4; DBD, DNA-binding domain; PRD, proline-rich domain; STY, serine–threonine–tyrosine domains; VEGF-A165, vascular endothelial growth factorA165, down syndrome candidate region 1 isoform 1 (DSCR1-1L). http://dx.doi.org/10.1016/j.abb.2015.02.003 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.

that TEF3-1 functions as an activator of down syndrome candidate region 1 isoform 1 (DSCR1-1L) in tumor angiogenesis [9–11]. Structurally, TEF3-1 contains an N-terminal TEA/ATTS domain, which is the most highly conserved DNA-binding domain (DBD) among all the TEAD members, and a C-terminal proline-rich domain (PRD) and serine–threonine–tyrosine (STY) domains [12,13]. A TEA domain is also found in the TEAD1 homologs AbaA (in Aspergillus) [13–15], TEC1 (in Saccharomyces cerevisiae), and scalloped (in Drosophila melanogaster) [1,16]. Most of the studies on TEF3-1 have been performed in mammalian cells such as muscle cells [17], endothelial cells [13] and embryos [18,19]. It has been recently discovered that TEAD family members interact with YAP in yeast cells specifically, the b loop and the a4-b10 motif of TEAD4 are shown to directly interact with YAP [20–22]. In addition, a role for TEAD and the Hippo-YAP pathway in tumorigenesis is proposed [22], but the underlying mechanism remains unclear. Taking advantage of the efficient reporter design of the yeast two-hybrid system, we cloned the human TEF3-1 sequence into pGBKT7 to express it in fusion with the Gal4 DNA-BD in yeast and explored the activation activity of TEF3-1 and its mutant forms on the reporter genes driven by UAS-containing promoters. Our results show that TEF3-1 and the two domains are sufficient to

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activate reporter genes in yeast cells, suggesting that human TEF31 can function as a transcriptional activator in yeast cells. Furthermore, the two domains of human TEF3-1 were also expressed in mammalian cells, to test if they are still active.

Table 1 Primers to construct the expression vector for TEF3-1 and the truncated sequences with pGBKT7 vector. Vector

Primers

TEF3-1

50 -ccggaattcttggagggca-30 (forward) 50 -cgcggatcctcattctttcac-30 (reverse)

Materials and methods muTEF3-1

Yeast strains, media and plasmids Yeast strain AH109, containing chromosomally integrated MEL1, ADE2, and HIS3 reporter genes, and plasmid pGBKT7, a 7.3 kb cloning vector that was used to generate bait proteins in fusion with GAL4 DNA-BD, were purchased from Invitrogen. Minimal SD Base and DO Supplements -Trp/-Leu/-His and -Trp and -His were obtained from Clontech. Salmon sperm DNA was obtained from Sigma and X-gal was obtained from Inalco. Mouse monoclonal antibody against Flag was purchased from Sigma– Aldrich (St. Louis, MO). Rabbit polyclonal antibody to down syndrome candidate region 1 isoform 1 was purchased from avivasysbio company (San Diego, CA).

50 -cgcggatcctcattctttcac-30 (reverse) TEF3-11–66

50 -ccggaattcttggagggcacgg-30 (forward) 50 - cgcggatcctcagattttgcgcct-30 (reverse)

TEF3-167–197

50 -ccggaattcatcctgtcgga-30 (forward) 50 - cgcggatcctcaaaaccctggcag-30 (reverse)

TEF3-1197–434

50 -ccggaattcgagtctcctgc-30 (forward) 50 -cgcggatcctcattctttcac-30 (reverse)

TEF3-1265–434

50 -ccggaattcgtggacatcc-30 (forward) 50 -cgcggatcctcattctttcac-30 (reverse)

TEF3-1301–434

50 -ccggaattcgacctcaacac-30 (forward) 50 -cgcggatcctcattctttcac-30 (reverse)

TEF3-1332–434

50 -ccggaattcacgaaggtctg-30 (forward) 50 -cgcggatcctcattctttcac-30 (reverse)

Cell culture and lentiviral expression TEF3-1197–265

Human umbilical endothelial cells (HUVEC) were obtained from Clonetics (San Diego, CA). Cells were grown on plates coated with 30 lg/ml vitrogen in EGM-MV BulletKit (5% FBS in EBM medium with 12 lg/ml BBE, 1 lg/ml hydrocortisone, 1 ll/ml GA-1000). HUVECs that were 70% confluent were used for most experiments. Cells were serum-starved in 0.1% FBS in EBM for 24 h prior to testing. Then the cells were cultured and transduced with lentiviruses carrying various genes, as previously described. Briefly, the lentiviral vector was generated in HEK 293T cells by FuGENE-6 HD transfection reagent (Roche Diagnostics, Indianapolis, IN) mediated transfection of the three plasmids. HEK 293T cells (5  106) were transfected with 5 lg of the transfer plasmid encoding the GFP gene, 1.25 lg pMD2G plasmid and 3.75 lg psPAX2 plasmid. Medium was removed approximately 14–16 h post-transfection, and refreshed with 10 ml of pre-warmed virus collecting medium. Medium was collected on days 2 and 3 posttransfection. The supernatant was separated by centrifugation (5 min at 7000g), filtered through a membrane with a 0.45 lm pore size. Titer of EGFP expressing lentiviral vector was determined by infection of HUVEC cells using serial dilutions in a six-well plate.

50 -ccggaattcaattggagggca-30 (forward)

50 -ccggaattcgagtctcctgc-30 (forward) 50 -cgcggatcctcacacggcttcga-30 (reverse)

Forward primers contain EcoRI recognition sites as underlined at its 50 -end and reverse primers contain BamHI recognition sites as underlined at its 50 -end.

Western blot analysis The yeast cells were centrifuged at 1500g for 5 min at 4 °C. Supernatants were decanted and the pellets were suspended in complete cracking buffer (8 M urea, 5% SDS, 40 mM Tris–HCl [pH 6.8], 0.1 mM EDTA, 50 mM PMSF, 0.02 mM Lepeptin, 0.005 mM Aprotinin, and 0.001 mM Pepstatin A) and lysed by ultrasonication. The resulting solutions were centrifuged at 13,000g for 5 min at room temperature. Supernatants were heated at 95 °C for 5 min and retained as yeast extracts, and total protein concentrations were determined. Western blotting was carried out as previously described [23].

Plasmid construction The plasmid expressing TEF3-1 fused to the GAL4 DNA-BD was constructed in pGBKT7 to be transfected into yeast cells. A mutant TEF3-1 plasmid, containing a two-base-pair shift before the start site of TEF3-1, was also constructed using pGBKT7. TEF3-1 truncation mutants were likewise created using pGBKT7. The primers used to create these truncation mutants all contain EcoRI or BamHI site and the sequences as shown in Table 1. In Table 2, TEF3-1 truncated fragments were inserted into pHAGE vector using BamHI and Xho I recognition sites.

Table 2 Primers to construct the expression vector for TEF3-1 and the truncated sequences with pHAGE vector. Fragments

Primers

TEF3-1

50 -ccgggatccttggagggcacggccgg-30 (forward) 50 -cgcctcgagtcattctttcaccagcctgtaga-30 (reverse)

TEF3-11–66

50 -ccgggatccttggagggcacg-30 (forward) 50 -cgcctcgagtcagattttgcgcct-30 (reverse)

TEF3-167–196

50 -ccgggatccatcctgtcggacg-30 (forward) 50 -cgcctcgagtcaaaaccctggca-30 (reverse)

LiAc-mediated yeast transformation

TEF3-1197–265

50 -ccgggatccgagtctcctgcag-30 (forward) 50 -cgcctcgagtcacacggcttc-30 (reverse)

In the LiAc transformation method, competent yeast cells are prepared and suspended in LiAc solution along with the plasmid DNA to be transformed. The denatured salmon sperm DNA are used during transformation as a carrier DNA to increase the transformation efficiency.

TEF3-1197–434

50 -ccgggatccgagtctcctgcagg-30 (forward) 50 -cgcctcgagtcattctttcaccagc-30 (reverse)

Forward primers contain BamHI recognition sites as underlined at its 50 -end and reverse primers contain Xho I recognition sites as underlined at its 50 -end.

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b-Galactosidase assays

Transwell migration assay

The filter assay was used to detect the transcriptional activity domain of TEF3-1. Briefly, fresh AH109 strains containing the various pGBKT7 constructs were transferred to SD/-Trp plates at 30 °C for 48 h, and then yeast cells were printed on the filter. The filter assays use at least one freeze/thaw cycle in liquid nitrogen to lyse the yeast cell walls. The procedures for the colony-lift filter were described in CLONTECH yeast protocols, which quantify b-galactosidase activity.

Serum-starved HUVEC with or without transduction with various lentiviruses as indicated were washed two times with PBS and incubated with 4 ml collagenase solution (0.2 mg/mL collagenase, 0.2 mg/mL soybean trypsin inhibitor, 1 mg/mL BSA, 2 mM EDTA in PBS) at 37 °C for 20 min. Cells were detached by gentle scraping and centrifuged at 1100 rpm for 3 min and washed twice with EBM containing 1% BSA, and seeded (1  105 cells/well) into the transwells coated with vitrogen (30 lg/mL) and inserted in 24-well plate wells containing 1 ml of the same medium. Then the cells were incubated at 37 °C for 1 h to allow the cell adhesion, and VEGF-A165 was added to the bottom well to a final concentration of 10 ng/ml. After additional 12 h incubation, cells migrated to the other surface of the membrane were fixed with 4% formaldehyde for 20 min, stained with 0.5% crystal violet for 15 min. The images were observed using microscope TS-100 (Nikon, Japan) and the cell numbers were counted. Data are expressed as the mean ± SE of quadruplicate values.

Molecular modeling The ExPASy http://swissmodel.expasy.org/ and MolProbity http://molprobity.biochem.duke.edu/ web servers were used for visualization of 3D protein structures. Actin staining Confluent HUVEC with or without transduction were stimulated with VEGF-A165 (vascular endothelial growth factor-A165) (10 ng/mL) for 20 min and washed twice with PBS, fixed with 4% paraformaldehyde/PBS for 10 min, washed in PBS 3 times with 5 min each, permeablized with 1% Triton X-100/PBS for 3–5 min, washed with PBS 3 times, blocked with 1% BSA for 30 min and incubated with rhodamine–phalloidin diluted in PBS (Molecular Probes/Invitrogen, Eugene, OR) for 30 min at room temperature, washed with PBS twice, and mounted with vectashield mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA). Cells were photographed under a Leica TCS SP1 confocal laser scanning microscope. Proliferation assay The effects of TEF3-1 and the truncated fragments on cell proliferation were characterized by cell counting. Briefly, after seeding 0.5  104 cells/well into a 6-well tissue culture plate for 24 h, HUVECs were transfected with different lentivirus. And 48 h later, the serum-starved medium contained 1% BSA in EBM with or without VEGF (10 ng/mL) was changed. Finally, after trypan blue staining, the cells were counted using a hemocytometer.

Statistical analysis The results are expressed as the mean ± S.E. variance. The Tukey–Kramer multiple comparisons test was used to determine statistical significance. A P value of

Characterization of the transcriptional activation domains of human TEF3-1 (transcription enhancer factor 3 isoform 1).

TEF3-1 (transcription enhancer factor 3 isoform 1) is a human transcriptional factor, which has a N-terminal TEA/ATTS domain supposedly for DNA bindin...
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