Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp

Abstract

NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp. We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain. We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.

Background

NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp.

Results

We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain.

Conclusions

We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.

Background

].

].

]. The C-terminal regions of NFAT proteins are unique in sequence and may be responsible for functional differences among NFAT family members.

HA-NFATp was purified by DNA affinity chromatography from baculovirus-infected Hi-five cell extracts. Portions of insect cell extract containing over-expressed HA-NFATp (lane 1), depleted extract (lane 2), and the purified/eluted HA-NFATp (lane 3) were resolved by SDS-PAGE and stained with coomassie brilliant blue. The position of full-length HA-NFATp is indicated with an arrow.

].

To date, biochemical analysis of NFATp and other NFAT members has been conducted using only portions and not the full-length versions of these proteins. Recovery of smaller, soluble, truncated forms of NFAT proteins is feasible relative to the more difficult task of isolating active forms of full-length NFAT proteins. Although such domain-based investigations have been constructive in studying the activity and the properties of the NFAT DNA-binding domains, a biochemical analysis of transcriptional activation and regulated nuclear association of NFAT necessitated the characterization of full-length proteins. This is mainly because the domains exhibiting the transactivation properties and those controlling nuclear association are located in regions flanking the central DNA binding domain. We therefore established protocols for expressing and purifying full-length recombinant human NFATp. Utilizing a reconstituted transcription system, we found that NFATp is a bona fide transcriptional activator and that activation requires regions of NFATp outside the DNA binding domain. In addition, we discovered that human NFATp produced in insect cells using a recombinant baculovirus is a phosphoprotein that demonstrates calcineurin-regulated association with nuclei in vitro.

Expression and purification of N-terminally tagged NFATp

).

HA-NFATp binds DNA alone and cooperatively with cJun/cFos

and data not shown) we conclude that recombinant HA-NFATp binds DNA both on its own and in cooperation with AP-1 proteins (cJun/cFos and cJun/cJun).

HA-NFATp binds DNA alone and cooperatively with cJun/cFos. DNase I footprinting assays were performed to investigate the binding of HA-NFATp to a region of the human IL-2 promoter in the absence (lanes 1-5) and presence (lanes 6-10) of recombinant human cJun/cFos (3.2 nM). HA-NFATp was added to reactions at the following final concentrations: 3 nM, lanes 2 and 7; 9 nM, lanes 3 and 8; 27 nM, lanes 4 and 9; and 54 nM, lanes 5 and 10. Footprinting reactions were resolved by denaturing PAGE and analyzed with a Molecular Dynamics PhosphorImager. Positions relative to the transcriptional start site (+1) of the human IL-2 promoter are indicated on the left. Locations of the previously characterized high affinity -45 NFAT site and composite element are indicated on the right.

HA-NFATp activates transcription in a reconstituted in vitro system

).

-E1b-CAT. The transcription system consisted of highly purified recombinant and native general transcription factors as described in the text.

). This demonstrates that NFATp is a bona fide transcriptional activator.

Activation domains of NFATp are required for transcriptional activation in vitro

]. In this study, however, transcriptional activation by NFATp was not dependent on the identified transactivation domains. The minimal DNA-binding domain of NFATp was sufficient to stimulate reporter gene activation, presumably by recruitment of endogenous AP-1 components through cooperative DNA binding. It is not possible to assess the true transactivation function of NFAT proteins in reporter assays that use a promoter comprised of NFAT/AP-1 composite elements in cells with high levels of endogenous AP-1 proteins. Our finding that recombinant HA-NFATp activates transcription in a reconstituted transcription system provided compelling evidence that NFATp is a bona fide activator. If NFATp acted alone to stimulate transcription in the reconstituted transcription system then the activation we observed would be dependent on the activation domains of NFATp. We therefore applied in vitro biochemical methods for the characterization of NFATp functional domains.

, GST-NFATp(1-921) activated transcription, while GST-NFATp(391-583) did not activate transcription. Thus, regions outside of the minimal DNA binding domain were required for transcriptional activation in a reconstituted transcription system. Taken together, our results directly demonstrate that NFATp is a transcriptional activator.

GST-NFATp(1-921), but not GST-NFATp(391-583) activates transcription in a reconstituted transcription system. Transcription reactions were performed in a reconstituted RNA polymerase II transcription system using a DNA template consisting of three high affinity NFAT sites upstream of the adenovirus major late core promoter and a 200 bp G-less cassette. 40 ng of either GST-NFATp(1-921) and GST-NFATp(391-583) were added to reactions as indicated. Transcripts were purified, resolved by denaturing PAGE, and visualized with a PhosphorImager.

Recombinant HA-NFATp is phosphorylated and exhibits regulated association with nuclei in vitro

]. This was confirmed by performing mass spectrometry on peptides recovered from a tryptic digest of recombinant NFATp (W. Clements and J. Goodrich, unpublished data).

HA-NFATp associates with nuclei in vitro only after treatment with calcineurin. HA-NFATp that was pre-treated with calcineurin and CaM (lanes 5 and 6) or mock treated (lanes 3 and 4) was mixed with HeLa nuclei. A control reaction was performed without added HA-NFATp (lanes 1 and 2). After incubation, the nuclei were pelleted by centrifugation and washed. Protein in the pelleted nuclei fractions (P) or supernatant fractions (S) were resolved by SDS-PAGE and analyzed by protein immunoblotting with anti-HA antibody. The positions of phosphorylated HA-NFATp and dephosphorylated HA-NFATp are indicated. The position of a non-specific protein in HeLa nuclei that is recognized by the anti-HA antibody is indicated with an asterisk.

, the majority of phosphorylated HA-NFATp was found in the supernatant and not in the pellet, while approximately 50% of the dephosphorylated NFATp was found associated with nuclei. Moreover, when HA-NFATp(331-722), which was not highly phosphorylated, was tested in the nuclei-association assay it was primarily found in the pellet with the nuclei, regardless of whether it was treated with calcineurin or not (data not shown). Taken together these data lead us to conclude that the phosphorylation state of recombinant HA-NFATp can control its association with nuclei in vitro in a manner that is very similar to the regulation of nuclear association observed for NFAT proteins in cells.

Conclusions

We have found that human NFATp is a bona fide transcriptional activator and that regions outside of the central DNA binding domain are required for NFATp to activate transcription. Our experiments in a reconstituted transcription system lacking contaminating AP-1 proteins and using DNA templates with high affinity NFAT sites demonstrate that NFATp can function as a transcriptional activator on its own. We have also shown that recombinant NFATp purified from insect cells is a phosphoprotein that exhibits association with nuclei in vitro only after dephosphorylation by calcineurin. The recombinant full length and deletion mutants of human NFATp used in the studies described here as well as the methods that we have developed will be valuable for future biochemical studies of the function of NFATp in DNA binding, transcriptional activation, phosphorylation/dephosphorylation, and nuclear association.

Plasmids and baculoviruses

-NFATp with SgrA I and filling in the overhang by treatment with Klenow. After further digestion with EcoR I, the NFAT DNA fragment was isolated and ligated into the Sma I and EcoR I sites of pVL1393HAX. A pVL1392-HAX-NFATp(331-722) was made by subcloning a Nde I - Sma I fragment from pGEX-NFATp(331-722) (J. Goodrich, unpublished) into the Nde I and Sma I sites of pVL1392-HAX.

-NFATp(NdeI) for site-directed mutagenesis with two oligonucleotides: a unique in-frame ATG codon contained in an Nde I site was created in place of the codon for amino acid 390 using an oligonucleotide of sequence 5'-CATCTGCAGCATCCATATGACTGCATCCCTC-3' and a stop codon was created in the NFATp cDNA after amino acid 583 using an oligonucleotide of sequence 5'-GTTGAAAGACAATAAACAGAATTCTGCCTGGTCTATG-3'. In the second step, the Nde I EcoR I fragment from pBS-NFATp(391-583) was ligated into the Nde I and EcoR I sites of pVL1392-GST.

].

Baculovirus stocks were produced according to standard procedures by individually cotransfecting parental plasmids (described above) along with Baculogold DNA (PharmMingen) into Sf-9 insect cells.

Expression and purification of recombinant proteins

, 0.2 M NaCl, 0.1% NP-40, 0.2 mM PMSF, 1 μg/ml leupeptin, 1.4 μg/ml pepstatin A, and 1 mM DTT). The eluate was spun through a Millex-GV4 filter, frozen in liquid nitrogen, and stored at -80°C. A similar protocol was used for the expression and purification of HA-NFATp(1-722), HA-NFATp(331-921), and HA-NFATp(331-722).

).

Recombinant GST-NFAT proteins were purified from Hi-five cell extracts (48 hr post-baculovirus infection) by affinity chromatography using glutathione-conjugated sepharose beads. Cell lysates (prepared as described above) were incubated with glutathione sepharose beads for 2 hours at 4°C. This was followed by extensive washes in TGEMD (1.0 M and 0.2 M NaCl). The purified proteins were eluted with reduced glutathione at 1 mg/ml in elution buffer (100 mM Tris (pH 7.9), 120 mM NaCl). The eluted fractions were spun through 0.22 μM Millex filters (Millipore), dialyzed for 6 hours at 4°C in dialysis buffer (20 mM Tris (pH 7.9), 100 mM KCl, 20% (v/v) glycerol, and 1 mM DTT), frozen in liquid nitrogen, and stored at -80°C. The expression and purification of recombinant human cJun/cFos (full length proteins) will be described elsewhere (H. Ferguson and J. Goodrich, manuscript in preparation).

DNase I footprinting

was added to each reaction. After a 30 sec incubation at 30°C, reactions were stopped with 40 μl of stop solution containing 25 mM EDTA, 125 mM KCl, and 10 μg of carrier yeast RNA. SDS was added to each reaction to a final concentration of 0.5%. Reactions were incubated at 65°C for 15 min and then placed on ice for 10 min. After a 10 min spin at 14,000 rpm in a microcentrifuge, the supernatants were transferred to new tubes. DNA was ethanol precipitated and resuspended in formamide loading buffer. Products were resolved by 8% denaturing PAGE.

In vitro transcription

].

Electrophoretic mobility shift assays

, 10 μg/ml calf thymus DNA, and 1.25 fmoles of double-stranded oligonucleotide bearing a high-affinity NFAT site (murine IL-4 promoter from -82 to -64). GST-NFATp(1-921) and GST-NFATp(391-583) were incubated with the template for 20 min at 30°C. Protein/DNA complexes and free DNA were resolved on 4% native polyacrylamide gels in 0.5X TBE for 4 hours at 4°C. Gels were dried on 3 mm chromatography paper (Whatman) and subjected to PhosphorImager analysis.

Dephosphorylation of recombinant NFATp proteins

. Control reactions were performed with NFATp proteins in the absence of added calcineurin and calmodulin. All reactions were incubated at 30°C for 20 min. Products were resolved on and 8% SDS gel and visualized by silver staining.

Nuclei association assays

, 10 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF). For each binding reaction, 5 μl of NFATp dephosphorylation reaction (or a mock treated control reaction) was added to 10 μl of nuclei slurry and allowed to incubate with gentle mixing for 10 min at room temperature. After incubation, nuclei were pelleted by centrifugation in a microfuge at 3000 rpm for 2 min. Supernatants were transferred to new tubes and nuclei were subsequently washed two times with 50 μl of buffer A. Protein in the supernatant and pellet fractions were dissolved in SDS sample buffer, resolved by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-HA antibody.

Acknowledgments

We thank Tim Hoey for the NFATp cDNA. J.A.G. is grateful to Robert Tjian and Tom Cech for their generous support, especially during the early stages of this work. This research was supported by a Public Health Service grant GM-55235 from the National Institutes of Health. J.A.G. is currently a Pew Scholar in the Biomedical Sciences and was a Special Fellow of the Leukemia Society of America during the early stages of this work. L.J.K. was supported in part by a NIH Predoctoral Training Grant T32 GM08345 and Beverly Sears Dean's Small Grants (University of Colorado).