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Gene Transcription after T Cell ActivationMolecular Regulation of Calcium-Stimulated CRACking the Code without Rosetta:

Mark Boothby

http://www.jimmunol.org/content/185/9/4969doi: 10.4049/jimmunol.1090097

2010; 185:4969-4971; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/185/9/4969.full#ref-list-1

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CRACking the Code without Rosetta: MolecularRegulation of Calcium-Stimulated Gene Transcriptionafter T Cell ActivationMark Boothby

After a “Big Bang” (1, 2) begat an ever-expandinguniverse of identified genes (CD Ags, cytokines,and so on) and insight into the structure of these

genes, a pressing need in immunology was to understand howchanges in gene expression are regulated during immuneresponses. For this purpose, the promoter regulation modelof Monod and Jacob (3, 4) and other lessons from the micro-bial world provided a compelling paradigm, that is, thatsequence-specific DNA-binding proteins would provide ananswer (5–7). Moreover, in the halcyon times of the 1980s,an organizing event for regulation of adaptive immunity wasthought to be the activation of a T lymphocyte through en-gagement of its Ag receptor. Indeed, it became clear that acomplex pattern of changes in mRNA levels followed T cellactivation. Accordingly, an urgent challenge was to under-stand what DNA-binding proteins (direct regulators of geneexpression, it was hoped) might be involved in controllinggenes after Ag receptor stimulation and to determine howthey would change their levels in the T cell nucleus afterlymphocyte activation.

Unlike in bacteria and viruses, however, the analyses of pro-moter mutations in eukaryotic cells relied on generation andsomewhat cumbersome molecular mutagenesis of promoters inreporter constructs. For mammalian systems, random and tar-geted mutagenesis of the germ line and screening to identifyregulatory proteins were, at best, pipe dreams somewhere on thehorizon, not thewidespread realities they are today. The Crabtreelaboratory’s publication of a Pillars article on this subject (8) wasimmediately recognizable as a major contribution and a big steptoward the broader goal of using T cell activation as a model forunderstanding TCR-stimulated mechanisms controlling entireprograms of gene expression. What was less clear at the outsetwas the breadth of the contribution in areas outside T cell biol-ogy and how central the protein identified as nuclear factor ofactivated T cells (NFAT) would become to our thinking aboutcalcium-regulated gene transcription.

With necessity and passionate curiosity being the mothersof invention, one simple solution to this need to understandtrans-acting regulators of gene expression was developed inthe form of the EMSA (9). Thus, one could end label specificpieces of DNA, mix them with the complex solutions yieldedby extracting nuclei for the nucleoplasm, and look for a changein migration after electrophoresis on non-denaturing gels. Inthe presence of nucleic acids designed to saturate associationsof proteins binding to DNA, almost any loosely associatedchromatin protein could be detected. Such assays screeningfor sequence-specific DNA-binding proteins (10) lent them-selves readily to comparisons of multiple cell types and cellsstimulated in various ways and analyzed across time courses.In addition, specificity controls using DNA containing theprobe sequence or point mutations compared with the probesequence and more refined “footprint” analyses (11) could bedeployed to establish with confidence and good moleculardetail the site(s) within a regulatory element with which theprotein(s) in a nuclear extract interact. In the in vitro foot-print analyses, one strand of a several hundred base pair pro-moter fragment was end labeled on one strand, and the posi-tions of phosphodiester bonds relatively protected from scissionwere identified on DNA sequencing gels. These analyses en-hanced the specificity and precision of localizing the sites rec-ognized by proteins present in the nuclear extract. Simple com-parisons of a resting and stimulated T cell line, Jurkat, and ofthis line to lines representative of other cell types, identifieda rapidly induced protein that specifically recognized one ofthe functionally mapped Ag receptor-responsive cis elementsin the IL-2 promoter. The mark of an important discovery isits influence on later research. What did this simple findingyield?

A first impact of this work was on understanding, at alevel of molecular mechanisms, the ways by which immuno-suppressant drugs could work. Induction of NFAT was soonfound to be blocked by the drug cyclosporin A (12) and tobe triggered by a calcium signaling pathway known to be cen-tral to T cell activation (13, 14). These two themes were linkedby beautiful insights into the mechanism of action of the im-munosuppressant cyclosporin A, showing that it acted in acomplex with an endogenous cellular protein of entirely un-related function to generate a unique molecular surface thatinhibits the calcium-regulated serine-threonine phosphatasecalcineurin (15). Thus, cyclosporin on its own, in the absenceof cyclophilin A, is unable to inhibit the calcineurin-mediateddephosphorylation of NFAT to allow release of the transcrip-tion factor from phosphorylation-dependent retention in the

Department of Microbiology and Immunology, Vanderbilt University School of Med-icine, Nashville, TN 37232

Address correspondence and reprint requests to Dr. Mark Boothby, Department ofMicrobiology and Immunology, Vanderbilt University School of Medicine, A-5301MCN, 1161 21st Avenue South, Nashville, TN 37232-2363. E-mail address: mark.boothby@vanderbilt.edu

Abbreviations used in this paper: NFAT, nuclear factor of activated T cells; Treg,regulatory T cell.

Copyright � 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

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cytosol. Conversely, on their own neither cyclophilin nor itspeptidyl prolyl isomerase activity regulates calcineurin orNFAT.

Another immediate impact of this Pillars paper was thatof identifying a high-priority target for molecular cloning.cDNAs encoding NFAT(s) turned out to be quite difficultto identify because the easiest methods that evolved duringthis era did not work, but in time, first one NFAT and thena family of NFAT proteins emerged (16, 17). These cloningefforts were the essential basis for gene targeting studies thatfirmly established the initial promise of NFAT as a TCR-induced regulator of immune function genes (18–21). Re-markably, though, these lines of work led to two furtherparadigm-shifting discoveries. First, a surprising finding wasthat a compound loss-of-function state for two members ofthe NFAT gene family led to unchecked inflammation andT cell hyperactivation (18, 22, 23). How could this be? Ul-timately, the paradox could be interpreted in the context ofthe rise of the suppressor cell, that is, dominant toleranceeffected by natural regulatory T cells (Tregs). Second, NFATand the relationship of its activation to SOCE (store-operatedcalcium entry) were brilliantly exploited as part of the ground-breaking work identifying Orai1 (24) as an essential compo-nent of SOCE (along with STIM1 and STIM2) and leadingto identification of an essential role for this process in de-velopment of natural Tregs (25, 26).

During the era when the universe of transcription factorswas young and undergoing explosive expansion, two themeswere woven into investigations. The first was that transcrip-tional function is built from the combinatorial interaction ofseveral proteins, for instance, at adjacent cis-acting elements.In this respect, a germinal contribution was the identificationof a crucial interaction between NFAT and dimeric proteinsof an AP-1 family of transcription factors. This insight wasa progenitor of a fundamental advance in thinking aboutunresponsiveness of conventional T cells, especially helper-type CD4 T lymphocytes (27). In particular, clonal anergy(28) can now be viewed as a consequence of nuclear NFAT,arising from sustained low-level calcium signaling in tolerizedlymphocytes, when it lacks an AP-1 partner and thereforedrives expression of a suite of genes differing from those ofnormal T cell activation (29). This paradigm, together withfindings about the role of NFAT in suppressive Treg devel-opment, is valuable in the context of downsides to the chronicuse of cyclosporin A, that is, its enhancement of a chronicvasculopathic effect.

A second theme integral to investigations of DNA-bindingtranscription factors is that a particular mobility shift comp-lex, or eukaryotic transcription factor, must necessarily regu-late far more genes than the one at which its function was firstimplicated. Thus, the identification of NFAT as an immedi-ate-early factor induced by T cell activation to trans-activateIL-2 gene transcription was followed by a stream of evidenceshowing that NFAT regulates scores of other genes in the ac-tivated T cell, including those encoding effector cytokines suchas IL-4 and IFN-g. Inasmuch as the effect on immunity ofchronically suppressing IL-2 production became a more com-plex picture, these diverse effects on effectors of immunity orallograft rejection add in important ways to the understand-ing of how calcineurin phosphatase inhibition prolongs graftsurvival.

There are certain periods when an explosion of foundatio-nal insights in an area of science can seem like the efflorescencethat moved physics in one memorable era or painting in an-other. Considering this Pillars paper together with a previousPillars article on the identification of NF-kB (10) and parallelwork of the time suggests that the latter half of the 1980s wassuch a period. Each of these articles is the progenitor of thou-sands of later papers, central to the identification of new ther-apeutic agents and avenues, and the sine qua non for how wethink about the regulatory networks that mediate effectiveimmunity while minimizing the risk of pathological autoim-munity. Some timeless lessons that bear repeating on a regularbasis are exemplified by this history. First, new technical toolscan change the way we think about a problem even when, inretrospect, they seem terribly primitive. Second, the data re-call an old truth that still applies in science: the perfect can bean enemy of the great. A number of details in each paperultimately were not true; NFAT is not T cell-specific, justas NF-kB is not B cell-specific. Imperfection notwithstand-ing, identification of this mobility shift complex became a pil-lar supporting an edifice of new knowledge not only aboutimmunology but also of important new insights into the path-ophysiology of cardiovascular diseases, cancer, bone and car-tilage biology, and more (30–35). Finally, then, this article isan important reminder of how work with a narrow initialfocus can have an unpredictable but immense cross-cuttingimpact on fields well beyond that of the initial study (in thiscase, immunology and T lymphocyte activation).

DisclosuresThe author has no financial conflicts of interest.

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