ETS transcription factor knockouts: A review

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Introduction Re ting peripheral blood T- ell remain qui ent for period of month to years until they en ounter the appropriate ognat peptid in the ontcxt of a ell- uri c major hi tocompatibili ty omplex (MH ) mole ul . T-ccll receptor (T R) binding of thi MHClpcptide comple initiate the proce ofT- II a tivation . A tivatcd T-cell undergo cries of highly rdin ted ch ngc in b th gene e pre ion and cellular proliferation.• 10 t notably. T-cell tivarion produ e the tran riptional tivation of hundred of novel gene including myri d of cytokine and cell- uri e re cptor . The e tran criptional event are yn hronized 10 hange in the cell y le, wh reby a tivated T-cell it Go and proliferate. The proce of 'l-cell a tivation i

mediated by ignal throughout the archite tur of the T- ell. I the cell urfa e. receptor uch a CD4. CD . and the T RlCD3 compl ti tc I of cell urf e iated and cytopl mic ignal tran du tion mol cule in luding tyro ine kina e ,. .2ras.' and al ineurin. The e cytopla mic mc eng rs, in turn, activate a set of Iran ripu on f .tors in luding ET ( 26-lran formation spe ific or 26pe ifi ) protein which are ultimate] re pon ible for the nu lear event regulating tivation- pe ific T-ccll gene .5 \ hile our identification of thc m leculc and biochemical event involved in T- ell a II at ion readily progre e . we till lack a d tailed undcr tanding of the fun tional ignific n c of the e interrelated pathway . In particular. we have yet 10 fully Identify the role of pccific tran npnon fa tor in regulating a tivati on-rclated

T-ccll gcn cxpre ion . For e ample. alth ugh we know that the regular ry I ment (pr rnotcr and enh n r ) of th IL-2.t> IL-2Ra. IL-3. and GM- f:'l gene all contain binding it for E'F tran cription fa t r . we do not full y understand the imponan e of individual ET pr oteins in h th po itivcly and n galively regulating these gene following T-ccll activation. Thus. our omplctc understand ing of hath nonnal and pathologi al T- ell fun lion require s the full elu idauon of the molecular me hani. ms that control tran: cription during T-cell activation . Dunng the pa I ten year . in estigator have e amined the mcchani m of a ' 11 vation-. pcci I ' T-c II gene e xprcs ' Ion \ 1.1 multiple di.tin ·t but mpl rncnt route.. Conunu don "(/' -12

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42 CLINICAL IMMUNOLOGY Newsletter

ETS Tranlcrlptlon Factor Knockout.: A Review (Continued from pg. 41)

Nuclear magnetic resonance (NMR) and X-ray crystallographic studies have provided us with the structures of several important T-cell transcription factors bound to DNA. Structure-function experiments have revealed the relevant transcription factor binding sites in the regulatory elements of activation specific T-cell genes . Biochemical studies have revealed the functional subdomains of proteins involved in T-cell specific gene expression and have identified the importance of post-translational events including phosphorylation and protein-protein interactions in regulating these transcription factors. Transgenic animals overexpressing individual wild-type or mutant transcription factors in T-cells have elucidated the relevance of these functional subdomains in vivo. Finally, several recent gene targeting studies producing knockout (gene targeted) cells and animals deficient in a particular transcription factor have begun to elucidate the roles of individual transcription factors in regulating immune cell development and function. These knockout experiments have the advantage over the previously mentioned investigative strategies in that they most definitively establish the obligatory function of a particular gene in the development and life of an organism. Using the multiple experimental approaches described above, many investigators have recently shown that the ETS family of transcription factors play important roles in T-cell activation, specifically,

and in immune cell development and function, generally. Most notably, a recent series of knockout experiments has firmly demonstrated the crucial role of ETS proteins in the vertebrate immune system . This review will: (i) briefly discuss current knockout technologies; (ii) provide an overview of the ETS family of transcription factors; and (iii) summarize our current understanding of the importance of specific ETS transcription factors (in T-cell activation specifically, and in immune cell development and function, generally) as derived from knockout experiments of individual ETS genes. Knockout Technologies Knockout mice are the product of a targeted mutation ablating the function of a particular gene. In vivo targeted mutagenesis relies on the phenomenon of homologous recombination, by which an exogenous gene (the targeting vector) inserted into a cell preferentially recombines with homologous endogenous sequences (the gene target). Such homologous recombination can be performed in murine embryonic stem cell lines (ES cells), which are totipotent stem cells .I~12 After targeting a gene on one chromosome of an ES cell, one can incorporate knockout ES cells into a murine blastocyst and subsequently implant them into pseudopregnant female mice. The mice that develop will be heterozygotic chimeras for the targeted mutation since some ofthe tissues will be derived from the ES cells. Since germ cells are haploid, some germ cells of these mice will contain chromosomes carrying only the tar-

geted mutation. If chimeric mice are mated with wild-type mice, and a germ cell containing the targeted mutation from the chimeric mouse fuses with a wild-type germ cell, all the cells of the developing embryo will be heterozygous for the gene disruption. Such heterozygotes can be mated to each other to produce mice homozygous for the targeted allele . These mice containing a homozygous null mutation for a particular gene are commonly referred to as "knockout mice." In addition to their use in producing knockout animals, ES cells can be differentiated in culture by various chemical and environmental manipulations to produce diverse cell types. 13,14 With regard to the immune system, ES cells have been shown to differentiate into multiple hematopoietic cell lineages.P''? Thus, ES cells containing targeted mutations of a particular gene in both chromosomes can be differentiated in cell culture to evaluate the functional importance of a gene during in vitro hematopoiesis. This in vitro application of knockout technology is particularly useful for mutations which cause early embryonic lethality in knockout zygotes . With the in vitro knockout ES differentiation assay, we can establish the obligatory function of a particular gene during ontogeny in cell culture even if the mice deficient in this gene are non-viable. The recombination activating gene-2 (RAG·2) deficient blastocyst complementation system represents an alternative knockout strategy employed for the in vivo evaluation of the functional importance of individual gene products in Tand B-Iymphocytes. IS Mice homozygous

CLINICAL IMMUNOLOGY NEWSLE1TIR (ISSN 0197-1859) is issued monthly in one indexed volume per yearby Elsevier Science Inc.• 6S5Avenue of theAmericas. NewYork. NY 10010. Subscription price per year: 5260 (DO. S12) for institutional subscribers. $232 (DO. 4S7) for personal subscribers. Both of these include postage and handling. Periodical postage paid at NewYork. NY, and at additional mailing offices. Postmaster: Sendaddress changes to Clinicallmmunolog-y Newslette« Elsevier Science Inc.• 6SSAvenue of theAmericas. NewYork. NY10010. NO'ffi: No responsibility is assumed by the Publisher for any injury and/or damage 10 persons or property as a matter of products liabilitj; negligence or otherwise. or fromany use or operation of any met~. products. ins~ctions or ideascontained in the material herein. Nosuggested testor procedure should becarried outunless. in thereader's judgment. itsriskis justified. Because of rapid adv~s In the medical sciences. we recommend thatthe independent verification of diagnoses and drugdoses should be made. Discussions. viewsand recommendations as to medical procedures. choice of drugs anddrugdosages are the responsibility of the authors.

0197-1859/99 (see frontmatter)

C 1999 Elsevier Science Inc.

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for a mutation in RAG-2 cannot produce mature B- or T-cells since they cannot initiate VDJ recombination.P''" All other cell lineages of RAG-2 deficient mice are capable of developing normally. 15 Injection of wild-type (untargeted) ES cells into RAG-2 deficient blastocysts produces chimeric mice which develop normal B- and T-cells, all of which are necessarily derived from the donor wildtype ES cells. By using homozygous targeted ES cells in place of wild-type cells, one can thus evaluate the functional importance of a targeted gene by assaying for the presence and function of lymphocytes. In these experiments, the presence of any lymphocyte subsets suggests that the targeted gene is not strictly required for the development of those cell types. Conversely, the absence of a particular lymphocyte subset establishes the obligatory function of the specific gene during development Similarly, functional defects (e.g., failure ofT-lymphocytes to proliferate in response to appropriate TCR stimulation) implicate the targeted gene product as an essential component of a particular pathway. Conversely, normal functioning demonstrates that the targeted gene is not strictly required in the relevant pathway. The RAG-2 deficient blastocyst complementation system, like the in vitro ES cell differentiation system, is particularly informative in the evaluation of mutations of essential gene products since the lymphocyte phenotypes of these mice can be evaluated even if the mutation is lethal in the traditional knockout system.

groove contacts occurring between the third alpha helix and the DNA. The ETS domains of the ETS proteins bind sequence motifs with the core sequence GGA. 20 ETS transcription factors have been highly conserved throughout evolution, playing important roles in organisms as phylogenetically diverse as worms." flies,26,27 mice, and humans.l In vertebrates, over 19 ETS proteins have been cloned to date including ETS -I, ETS-2, ELF-I, ELF-2, ELF-3, PU.l, SPI-B, FLI-I , GABPa, ELK-I, ERM, TEL, ERF, ERG , PEA3, SAP-I, SAP-2, ER81 , and ER71. While these ETS proteins are expressed in a variety of tissues, many of these ETS factors are preferentially expressed in immune cell lineages. Moreover, ETS proteins have been implicated as important regulators of immune cell development and function. In T-cells specifically, ETS binding sites have been identified in the transcriptional regulatory elements of a number of major genes including IL-2,6 IL-2Ra,7 IL-3,8 GM-CSF,28CD4,29and TCRa. 21 ETS proteins can be segregated into sub-families based on the structural differences between their DNA binding domains.P:" These ETS subfamilies possess subtle differences in their DNA binding specificities in that they recognize the core GGA sequence only in the presence of particular flanking nucleotides. For the purpose of this review, it is important to note that ETS-I and ETS-2 belong to one subfamily, PU.l and SPI-B make up a different subfamily, and TEL belongs to a third distinct subfamily of ETS proteins.

The ETS Family of Transcription

Factors: an Overview

ETS-l

The ETS family of nuclear proteins was originally identified by sequence homology with the V-ETS proto-oncogene of the avian retrovirus, E26. 18.19 In chickens, the GAG-MYB-ETS fusion protein is oncogenic, causing erythroblastosis and myeloblastosis. The erythroblastic property of the E26 virus depends on an intact ETS protein. ETS proteins are sequence-specific transcription factors. 20,21 Each ETS family member shares a related 82 amino acid DNA binding domain (the ETS domain) that has been shown by NMR22.2J and X-ray crystallography" to have a winged helix-loop-helix conformation, with major

The ETS-I gene was originally discovered by sequence similarity with the avian E26 oncovirus.P:'" ETS-l is predominantly expressed in the immune cells of chickens, mice, and humans. 32•35 Several posttranslational modifications appear to regulate the function of the ETS-I protein. These include intramolecular inhibition of DNA binding,36'38 phosphorylationdependent inhibiton of DNA binding,39-41 Ras-dependent phosphorylation and activation of the transactivation domain.f and numerous protein-protein interactions.' Functionally important ETS -I binding sites have been identified in the regulatory elements of a number of important 10 1999 Elsevier Science Inc.

immune cell genes including the TCRa enhancer,2J.43 the TCRB enhancer,8 .44.45 the IgH JlA and JlB enhancers," the IL-2RB promoter.f the lck promoter.P:" the MHCII DRA promoter,50.51 the CD8a intronic enhancer," the TdT promoter," and the HIV-I LTR.54-57

ETS-l Knockout Studies To assess the role of the ETS-I gene product in lymphocyte development and function, two teams of investigators independently produced homozygous ETS-l gene disrupted ES cells (ETS-I·1- cells) and employed these cells in the RAG-2 deficient blastocyst complementation system. 58,59 Analysis of RAG-2-1- ETS-I-1chimeric mice revealed a number of important lymphocyte anomalies. First, while all thymocyte subsets were present, total numbers of thymocytes were decreased. Second, there was an increased percentage of double negative (CD4'CD8') thymocytes and a decreased percentage of double positive (CD4+CD8+) thymocytes. Third, there was a decrease in the total number of lymph node and splenic T-cells. Moreover, these lymph node and splenic T-cells spontaneously apoptosed when cultured in the absence of activating signals. Fourth, the number of splenic IgM+1B220dull B-cells was increased. This increase correlated with an increased total number of splenic plasma cells and a fiveto ten-fold increase in serum IgM. Fifth, splenic T-cells failed to appropriately proliferate in response to activation by ConA or a-CD3 mAb. These experiments thus demonstrate that while ETS-I is not required for the development of mature T- and B-cells, ETS-I deficiency results in defective TCR mediated activation, decreased mature T-cells, increased splenic and lymph node B-cells, and increased serum IgM. Several models for the role of the ETS-I protein may be proposed from the lymphocytic phenotypes of the RAG-2·1. ETS-I·1chimeric mice . First, since T- and B-cell development occurs in the chimeras, either ETS-l is not required for the development of these lineages, or other ETS transcription factors can replace ETS-l in regulating the transcription of ETS-l responsive genes. Second, ETS-I expression may maintain mature T- and B-cells in a resting state. In this model, ETS-I

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deficiency results in spontaneous cell proliferation: T-cells progress through the cell cycle unchecked and apoptose, resulting in decreased mature T-ceIls, and B-cells differentiate into plasma cells, resulting in increased plasma cell numbers and increased serum IgM. Finally, ETS-I itself may regulate genes involved in apoptosis. In this model. ETS-l deficiency would result in apoptosis ofT-cells and decreased T-cell numbers. To study the role of ETS-I in cell lineages other than lymphocytes, knockout mice containing a null mutation of the ETS-I gene were produced." In addition to the lymphocytic defects found using the RAG-2 deficient blastocyst complementation system, several other anomalies were discovered. While ETS- I knockout mice were both viable and fertile . they displayed an increased perinatal mortality. with approximately 50% of the mutant pups dying before four weeks of age. The etiology of this premature mortality is still under investigation. Examination of various cell lineages in the ETS-I knockout mice revealed normal numbers of erythrocytes, monocytes, neutrophils, and megakaryocytes. However. ETS- I knockout mice contained significantly reduced splenic natural killer (NK) cells . Moreover, these mice displayed defective NK cell cytolytic activity against both tumor cetl and class I MHC-deficient targets. The defective NK function did not reflect defective expression of cytokine receptors important in NK cell function (IL-2R, IL15R, or IL-18R) and the NK defect could not be rescued by exogenous cytokines. Thus ETS-I appears to play an essential role in the development of NK cells.

PU.1 PU.I was originally identified as the product of the PU.I-SPI- I-SFPI-I protooncogene." PU .I expression is restricted almost exclusively to hematopoietic cell lineages. suggesting its important role in hematopoiesis.6z•65 PU.I contains a well defined N-terminal transactivation domain66,67 as well as a potentially proteasesensitive PEST region." Like ETS-I, PU.I is involved in several protein-protein interactions.' PU.I can be phosphorylated both in vitro and in vivo.68,69 This phosphorylation may occur via a JNKI dependent pathway." Functionally important

o191·18~9/99 (see frontmaner)

PU ,I binding sites have been described in the regulatory elements of several immune ceU-specific genes including the IgJ chain promoter." IgVld9 light chain promoter;" the CDI8/CDlla promoter,72.73 the FcyRI promoter," the IgH J.1A and J.1B enhancer elcments.46 the IgH enhancer 1t element.P:" the PU.I promoter, n the CD I0 promoter," the GM-CSF-Ra enhancer.I? the Igx E3' element,SO,sl the CDllb enhancer.'" IL-4 2nd intronic enhancer," and the Ig),. 2-43'

enhancer,"

PU.l Knockout Studies The production ofPU.l knockout mice has established a pivotal role for PU.I during hematopoiesis." PU.I knockout mice die between embryonic day 17 and day 18. These embryos produce normal numbers of erythrocyte progenitors, but they display an embryonic anemia as a result of impaired erythroblast maturation. While the PU.I knockout embryos produce normal numbers of megakaryocyte progenitors, they display severe defects in producing progenitors for granulocytes, monocytes, T-cetIs, and B-cells. This phenotype was also observed when the PU.I knockout fetal liver cells were adoptively transferred to lethally irradiated adult hosts. demonstrating that the hematopoietic defect was cell autonomous." Taken together, these results suggest an essential role for PU.I in the development ofT-celis. Bcells, and myeloid cells. The data suggest a model in which a PU .l-dependent pluripotent progenitor cell may give rise to both lymphoid and myeloid lineages. The role of PU .I in myeloid ontogeny was further evaluated using the PU.I knockout ES cells in the in vitro ES knockout cell differentiation system,87.88 and by differentiating the myeloid cells of the PU.I deficient embryos in vitro." Several markers of early myeloid development, including rnyeloperoxidase, GCSF, and GM-CSFR genes were expressed in the hematopoietic colonies derived from the PU.I knockout embryos and the PU.I knockout ES cells. However, markers of terminal myeloid differentiation, such as M-CSFR, CDllb, and CD64 were absent from both the PU .I knockout embryo and the P~J.l knockout ES cell derived hematopoietic colonies. The failure of the knockout PU.I ES cells to differentiate into macrophages was rescued 4:l 1999 Elsevier Science Inc.

by a PU.I transgene under the control of its own promoter. Taken together, these in vitro results suggest that while PU.I expression is not strictly required for early myeloid development, it is crucial for terminal myeloid differentiation.

SPI·B SPI-B was originally cloned from a Burkitt lymphoma cell library, using the ETS domain of PU.I as a probe." SPI-B expression is restricted to lymphocytes." In adult mice. SPI-B is expressed in the germinal centers of lymph nodes. the white pulp of the spleen, and the medulla of the thymus. The SPI-B protein has 67% amino acid identity with PU .I. 90 The transactivation domain of SPI-B is similar to that of PU.l .9/ Like PU .1. it can be phosphorylated in its PEST sequence.f" This phosphorylation can occur via both ERKI and JNKI dependent pathways/" The murine SPI-B and PU .I sequences share 70% identity in their ETS domains." and SPI-B has been shown to bind to all known PU .I target genes."

SPI·B Knockout Studies While SPI-B and PU.I share many characteristics, their different patterns of expression suggest that they may not play redundant roles in the immune system. Moreover, as described above, SPI -B cannot assume the function of PU.I in PU.I knockout mice. To identify the unique function of SPI-B in vivo, SPI-B knockout mice were produced." These SPI-B knockout mice are viable and fertile. Furthermore. these mice do not display abnormalities in the development of myeloid, erythroid, or lymphoid lineages. Functionally. SPI-B knockout T-cells are normal. In contrast, the B-cells of the SPI-B knockout animals display severe abnormalities in both functional and humoral responses. The B-Iymphocytes respond poorly to in vitro anti-IgM stimulation. In vivo, SPI-B knockout mice exhibit anomalous T-dependent responses . In particular, they produce decreased levels ofIgG I. IgG2a. and IgG2b, they form small germinal centers which cannot be sustained over a 21-day period. and they contain increased apoptotic splenic B-cells relative to control animals. These results demonstrate that SPI-B plays a distinct role from PU.I in immune celt

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development and that SPI-B is required for normal B-cell receptor-mediated events.

TEL The TEL gene was recently identified as a rearranged locus in the t(S;12) chromosomal translocation in human chorionic myelomonocyticleukemia.941EL rearrangements have since been found in several human leukemias. These rearrangements result in fusions with a number of different proteins. 9s-97 Most notably, the TEU AML-I fusion has been observed in 25 to 30% of all childhood acute lymphoblastic leukemias." TEL is expressed in a wide variety of normal mouse tissues, including heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis." The transcriptional properties, post-translational modifications, and in vivo targets of the TEL protein have not yet been described.

TEL Knockout Studies To determine the role of the TEL gene product during normal murine development TEL knockout mice were produced." Knockout TEL embryos died between embryonic day EIO.S and EI1.S. To determine if the hematopoietic progenitors in the TEL knockout embryos were capable of normal differentiation, yolk saks from the E9.5 embryos were differentiated in vitro. Normal numbers of erythroid, macrophage, and mixed colonies were observed upon the addition of appropriate cytokines. Thus, myeloerythroid hematopoiesis does not strictly require the TEL protein. Interestingly, while the TEL knockout yolk-saks were capable of hematopoiesis, these yolk-saks lack vitelline vessels. Other abnormalities in the TEL knockout embryos include abnormal apoptosis in mesenchymal and neural tissues. Our understanding of the requirement of the TEL transcription factor in normal lymphocytes awaits the introduction of the TEL knockout cells into the RAG-2 deficient blastocyst complementation system.

ConclusionIFuture Directions The above review of the ETS knockout studies published to date firmly establishes that ETS proteins perform diverse developmental and functional roles in a wide variety of tissues. In particular, the T-cell

CLINICAL IMMUNOLOGY Newsletter 45

abnormalities described in several of these reports confirm the hypotheses of hundreds of previous structural, molecular, genetic, and biochemical studies. By considering the knockout studies together, several novel conclusions emerge. First, the finding that all of the ETS knockout animals display phenotypic abnormalities proves that the ETS proteins cannot simply function redundantly (despite the fact that many of the different ETS proteins are expressed simultaneously in the same tissues). Second, the finding of abnormal apoptosis in the ETS-l, SPI-B, and TEL knockout animals suggests a general role for ETS proteins in regulating apoptosis in multiple cell lineages. Third, the vastly disparate phenotypes of the different ETS knockout mice clearly demonstrate that a complete elucidation of the biology of ETS transcription factors requires the cloning and subsequent production of knockouts of all of the family members. Fourth, it is important to note that ETS proteins do not act alone in regulating transcription. Specifically, in 'l-cells, ETS factors have been shown to physically interact with, and coordinately regulate gene expression with other families of transcription factors including the widely expressed AP-l, 100 NF-lCB, and NFAT proteins.'?' Many of these transcription factors have been gene targeted, and the phenotypes of the knocklO2- l3I . 1y stud'ed out mice have been extensive I and reviewed, 132-137 Our understanding of the molecular mechanisms that govern T-cell specific gene expression will be greatly facilitated by the characterization and cross breeding of these different knockout animals. Finally, it is clear that our understanding of the importance of ETS (and any other) transcription factor is limited by our present technologies. For example, in the case of an early embryonic lethal phenotype, the knockout technologies reviewed above do not allow us to assess the importance of the targeted gene product at a late stage of development and/or in a particular tissue. Thus, our complete understanding of the molecular mechanisms that govern the transcription ofT-lymphocytes (and any other cell lineage) awaits the continued application of present knockout technologies as well as future innovations including the routine production of developmentally timed and tissue-specific gene-targeted animals. Q 1999 Elsevier Science Inc.

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29. Wurster AL, Siu G. Leiden JM. Hedrick SM : Elf-I binds to a critical element in a second CD4 enhancer. Mol Cell Bioi 14:6452·6463. 1994.

42. Yang as, Hauser CA. Henkel G. Colman MS. Van Beveren C, Stacey KJ. Hume DA. Maki RA. Ostrowski MC : Ras-rnediated phosphorylation of a conserved threonine residue enhances the transactivation activities of cETSI and c·ETS2. Mol Cell Bioi 16:538-547, 1996.

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32. Bhat NK. Fisher RJ. Fujiwara S. Ascione R. Papas TS : Temporal and tissue-specific expression of mouse ETS genes . Proc Nat! Acad Sci USA 84:3161-3165,1987. 33. Bhat NK. Komschlies KL, Fujiwara S. Fisher RJ. Mathieson BJ. Gregorio TA. Young HA. Kasik JW. Ozato K. Papas TS : Expression of ETS genes in mouse T-thymocyte subsets and T-cells . J Immunol 142:672·678. 1989. 34 . Ghysdac:1 J. Gegonne A. Pognonec P. Demis D. Leprince D. Stehelin D: Identification and preferential expression in thymic and bursal lymphocytes of a c-ETS oncogene-encoded Mr 54.000 cytoplasmic protein . Proc Natl Acad Sci USA 83:1714-1718.1986. 35. Chen JH : The proto-oncogene c·ETS is preferentially expressed in lympho id cells . Mol Cell Bioi 5:2993·3000. 1985. 36. Lim F. Kraut N. Frampton J. GrafT: DNA binding by c·ETS-I. but not v·ETS. is repressed by an intramolecular mechanism. EMBO J II :643-652. 1992. 37. Hagman J. Grosschedl R: An inhibitory car boxyl-terminal domain in ETS·I and ETS·2 mediates differential binding of ETS family factors to promoter sequences of the mb-I gene. Proc Natl Acad Sci USA 89:8889-8893. 1992. 38. Hahn SL. Wasylyk B: The oncoprotein v·ETS is less selective in DNA binding than c·ETS·I due to the C·terminal sequence change . Oncogene 9 :2499-2512. 1994. 39. Fisher RJ. Mavrothalassitis G, Kondoh A, Papas TS : High-affinily DNA·protein interactions of the cellular ETS I prote in: the determination of the ETS binding motif. Oncogene

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45. Wotton D. Lake RA. Farr Cf, Owen MJ: The high mobility group transcription factor. SOX4. transactivates the human CD2 enhancer. J Bioi Chern 270 :7515-7522.1995. 46. Nelsen B. TIan G. Erman B. Gregoire J. Maki R. Graves B. Sen R: Regulation of lymphoidspecific immunoglobulin mu heavy chain gene enhancer by ETS·domain prote ins. Science 261 :82·86.1993. 47. Lin JX. Bhat NK. John S. Queale WS. Leonard WJ: Characterization of the human interleukin-Z receptor beta-chain gene promoter: regulation of promoter activity by ETS gene products. Mol Cell Bioi 13:6201-6210. 1993. 48 . Leung S. McCracken S. Ghysdael J, Miyamoto NG: Requirement of an ETS-binding element for transcription of the human lck type I promoter. Oncogene 8:989-997.1993. 49. McCracken S. Leung S, Bosselut R. Ghysdael J. Miyamoto NG : Myb and ETS related transcription factors are required for activ ity of the human Ick type I promoter. Oncogene 9:3609· 3615.1994. 50. Jabrane FN, Peterlin BM : ETS-I activates the DRA promoter in B-cells. Mol Cell Bioi 14:7314·7321.1994. 51. Peterlin BM: Transcriptional regulation of HLA-DRA gene . [Review). Res Immunol 142:393·399. 1991. 52 . Hambor JE, Mennone J. Coon ME, Hanke JH. Kavathas P: Identification and characterization of an Alu-containing, T-ce))·specific enhancer located in the last intron of the human CD8 alpha gene . Mol Cell Bioi 13:7056-7070. 1993.

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53. Ernst p. Hahm K. Smale ST: Both LyF-I and an ETS protein interact with a critical promoter element in the murine terminal transferase gene. Mol Cell BioI 13:2982-2992, 1993. 54. Holzmeister J. Ludewig B. Pauli G. Simon 0 : Sequence specific binding of the transcription factor c-ETS I to the human immunodeficiency virus type I long terminal repeat. Biochem Biophys Res Comm 197:1229-1233, 1993. 55. Pazin MJ. Sheridan PL, Cannon K. Cao Z. Keck JG. Kadonaga JT. Jones KA: NF-kappa B-mediated chromatin reconfiguration and transcriptional activation of the HIV-I enhancer in vitro. Genes DeveII0:37-49. 1996.

KouzaridesT: The activation domain of tran scription factor PU.l binds the retinoblastoma (RB) protein and the transcription factor TFIIB in vitro: RB shows sequence similarity to TFIID and TFIIB. Proc Natl Acad Sci USA 90:1580-1584.1993. 67. Klemsz MJ. Maki RA: Activation of transcription by PU.I requires both acidic and glutaminedomains. Mol CeI1 Bioi 16:390-397.1996. 68. Pongubala JM. Van BC. Nagulapalli 5, Klemsz MJ, McKercher SR. Maki RA, Atchison ML: Effect of PU.I phosphorylation on interactionwith NF-EM5and transcriptional activation. Science 259:1622-1625. 1993.

56. Seth A. Hodge DR. Thompson OM. Robinson L. Panayiotakis A, Watson OK, Papas T5 : ETS family proteins activate transcription from HIV-I long terminal repeat. AIDS Res Hum Retrovir9:1017-1023. 1993. 57. Sheridan PL. Sheline CT.Cannon K. Voz ML. Pazin MJ, Dadonaga IT. Jones KA: Activation of the HIV-I enhancer by the LEF-I HMG protein on nucleosome-assembled DNA in vitro. Genes Devel 9:2090-2104. 1995. 58. Borles JC, Wil1erford OM. Grevin D. Davidson L, Camus A. Martin P, Stehelin D. Alt FW: Increased T-cell apoptosis and terminal B-cell differentiation induced by inactivation of the ETS-I proto-oncogene. Nature 377:635-638. 1995. 59. Muthusamy N. Barton K. Leiden JM: Defective activation and survival ofT-cells lacking the ETS-I transcription factor. Nature 377:639-642, 1995. 60. Barton K. Muthusamy N. Fischer C. Ting CN, Walunas TL, Lanier LL. Leiden JM: The ETSI transcription factor is required for the development of natural killer cel1s in mice. Immunity 9:555-563. 1998. 61. Moreau GF: Spi-lIPU.I: an oncogene of the ETS family. [Review]. Biochimica et BiophysicaActa 1198:149-163.1994. 62. Hromas R, Orazi A, Neiman RS. Maid R. Van BC, Moore J. Klemsz M: Hematopoietic lineage- and stage-restricted expression of the ETS oncogene family member PU.1. Blood 82:2998-3004. 1993. 63. Klemsz MJ. McKercher SR. Celada A. Van BC. Maid RA: The macrophage and s-eenspecific transcription factor PU.l is related to the ETS oncogene [see comments]. Cel1 61:113-24,1990.

69. Mao C. Ray-Gallelt D. Tavitian A. MoreauGachelin F: Differential phosphorylationsof Spi-B and Spi-I transcription factors. Oncogene 12:863-873. 1996. 70. Shin MK, Koshland ME: ETS-related protein PU.I regulates expression of the immunoglobulin J-chain gene through 8 novel ETS-binding element. Genes DeveI7 :2006-2015. 1993. 71. Schwarzenbach H, Newell JW, Matthias P: Involvementof the ETS family factor PU.l in the activation of immunoglobulin promoters. J BioI Chern 270:898-907. 1995.

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64. Pettersson M. Sundstrom C. Nilsson K. Larsson LG: The hematopoietic transcription factor PU.I is downregulated in human multiple myeloma cell lines. Blood 86:2747-2753. 1995.

77. Kistler B. Pfisterer P.Wirth T: Lymphoid-and myeloid-specificactivity of the PU.I promoter is determined by the combinatorial action of octamer and ETS transcription factors. Oncogene 11 :1095-1\06. 1995.

65. Tenen DO: PU.I (Spi-1) and ClEBP alpha regulate expression of the granulocytemacrophage colony-stimulating factor receptor alpha gene. Mol Cell BioI 15:5830-5845, 1995. 66. Hagemeier C. Bannister A, Cook A.

78. lshimaru F. Shipp MA: Analysis of the human CD 100neutrai endopeptidase 24.11 promoter region: two separate regulatory elements. Blood 85:3199-3207. 1995. 79. Hohaus S. Petrovick M. VosoM. Sun Z. Zhang 0, Tenen 0 : PU.I (Spi-I) and CIEBPa

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regulate expression of the granulocytemacrophage colony-stimulating factor receptor a gene. Mol Cell Bioi 15:5830-5845, 1995. 80. Hirarnatsu R, Akagi K. Matsuoka M. Sakumi K, Nakamur H, Kingsbury L . Chella D. Hardy R, YamamuraK. Sakano H: The 3' enhancer region determines the Bff specificity and ProBlPre-B specificity of immunoglobulin VIC-JIC joining. Cell 83:1\ 13-1123,1995. 81. Pongubala J. Nagulapalli S. Klemsz M, McKercher S. Maki R. Atchison M: PU.I recruits a second nuclear factor to a site important for immunoglobulin IC 3' enhancer activity. Mol Cell Bioi 12:368-378. 1992. 82. Hickstein DO. Baker OM, Gollahon KA. Back AL: Identification of the promoter of the myelomonocytic leukocyte integrin CD II b. Proc Nat! Acad Sci USA 89:2105-2109.1992. 83. Henkel G. Brown MA: PU.I and GATA: components of a mast cell-specific interleukin 4 intronic enhancer. Prot Natl Acad Sci USA 91:7737-7741,1994. 84. Eisenbeis CP. Singh H. Storb U: PU.I is a component of a multiprotein complex which binds an essential site in the murine immunoglobulin lambda 2-4 enhancer. Mol Cell BioI 13:6452-6461,1993 . 85. Scott EW. Simon MC. Anastasi J. Singh H: Requirement of transcription factor PU.I in the development of multiple hematopoietic lineages. Science 265:1573-1577. 199.4. 86. SCOlt EW. Fisher RC. Olson MC, Kehrli EW, Simon MC, Singh H: PU.I functions in a cellautonomous manner to control the differentiation of multipolentiallymphoid-myelo id progenitors. Immunity 6:437-447,1997 . 87. Olson MC. Scott EW. Hack AA. Su GH, Tenen DO. Singh H. Simon MC: PU. I is not essential for early myeloid gene expression but is required for terminal myeloid differentiation. Immunity 3:703-714. 1995. 88. Henkel GW. McKercher SR. Yamamoto H. Anderson KL. Oshima RG, Maki RA: PU.I but not ETS-2 is essential for macrophage development from embryonic stem cells. Blood 88:2917-2926.1996. 89. Olson M. Scott E, Hack A, Su G. Tenen D. Singh H, Simon C: PU.I is not essential for early myeloid gene expression but is required for terminal myeloid differentiation, Immunity 3:703-714.1995. 90. Ray O. Bosselut R. Ghysdael J. Manei MG, TavitianA. Moreau GF: Characterization of Spi-B, a transcription factor related to the putative oncoprotein Spi-IIPU .I . Mol Cell Bioi 12:4297-4304, 1992. 91. Su G. Ip H. Cobb B. Lu M. Chen H, Simon C: The ETS protein Spi-B is expressed exclusively in B-cells and T-cells during development. J Exp Med. in press. 1996. 92. Chen H. Zhang P, Voso M. Hohaus S, Gonzalez D, Glass C. Zhang 0, Tenen 0 : Neutrophilsand monocytes express high

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levels of PU.I (Spi-l) but not Spi-B. Blood 8S:2918-2928. 1995.

93. Su GH. Chen HM. Muthusamy N. GarrenSinha LA. BaunochD, TenenDG. Simon MC: DefectiveB-eell receptor-mediated responses in mice lacking the ETS protein.Spi-B, EMBOI 16:71\8-7129, 1997. 94. GolubTR. BarkerGF, Lovett M. Gilliland DG: Fusion of POOFreceptorbeta to a novel ETS-likegene, tel. in chronic myelornonocytic leukemiawith t(S;12)chromosomal translocation. Cell 77:307-316. 1994. 9S. GolubTR, Goga A. BarkerGF. Afar DE. McLaughlin 1, BohlanderSK. RowleyID. WitteON. Gilliland DG: Oligomerization of the ABLtyrosinekinase by the ETS protein TEL in human leukemia. Mol Cell Bioi 16:4107-4116.1996. 96. RomanaSP,Mauchauffe M. Le Coniat M. Chumakov I. Le PaslierD. BergerR. Bernard OA: The t(l2;21) of acute lymphoblastic leukemiaresults in a tel-AMLI gene fusion. Blood 8S :3662-3670. 1995. 97. Buijs A. Sherr S. van Baal S. van BezouwS. van der Plas D. Geurts van Kessel A. Riegrnan P. Lekanne DeprezR. ZwarthoffE, Hagerneijer A et al.: Translocation (12;22) (p13:q11)in myeloproliferative disorders resultsin fusionof the ETS-likeTEL gene on 12pl3 to the MNI gene on 22qll [published erratumappears in Oncogene 11 :809, 1995]. Oncogene 10:1511-1519. 1995. 98. Rornana SP, Poirel H. LeconiatM. Flexor MA. Mauchauffe M. JonveauxP. Macintyre EA, BergerR, BernardOA: High frequency oft(l2:21) in childhood Bvlineage acute lymphoblastic leukemia Blood 86:4263-4269. 1995. 99. WangLC. KuoF. FujiwaraY. GillilandDG. GolubTR, Orkin SH: Yolk sac angiogenic defectand intra-embryonic apoptosisin mice lacking the ETS-related factorTEL. EMBO1 16:4374-4383, 1997. 100. BassukAG. Leiden1M:A direct physical association betweenETS and AP-I transcription factors in normalhumanT-cells. Immunity 3:223-237. 1995. 101. BassukAG.Anandappa RT. LeidenJM: Physical interactions betweenETS and NFkappaBINFAT proteinsplay an important role in their cooperative activation of the human immunodeficiency virus enhancerin T-cells. 1 ViroI71:3S63-3573. 1997. 102. Hu E. MuellerE. OlivieroS, Papaioannou va JohnsonR. Spiegelman BM: Targeted disruption of the c-fos gene demonstrates c-fosdependent and fos-independent pathways for gene expression stimulatedby growth factors or oncogenes. EMBO1 13:3094-3103, 1994. 103. OkadaS. WangZQ. Grigoriadis AE. Wagner EF.von RudenT: Mice lackingc-fos have normalhematopoietic stem cells but exhibit alteredB-cell differentiation due to an impaired bone marrowenvironment. Mol Cell BioI 14:382-390.1994.

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104. Hilberg F, Aguzzi A. Howells N. WagnerEF: C-jUD is essential for normalmousedevelopmentand hepatogenesis [published erratum appears in Nature 366:368, 1993]. Nature 365:179-181 , 1993. lOS. HilbergF. Wagner EF: Embryonic stem(ES) cells lackingfunctional c-jun:consequences for growthand differentiation. AP-l activity and tumorigenicity. Oncogene 7:2371-2380, 1992. 106. Field sr, Johnson RS. Mortensen RM. Papaioannou VE. Spiegelman 8M. Greenberg ME: Growth and differentiation of embryonic stemcells that lack an intact c-fosgene. Proc NatlAcad Sci USA89:9306-9310. 1992. 107. Wang ZQ. Ovitt C. Grigoriadis AE. MohleSteinlein U. RutherU. Wagner EF: Boneand haematopoietic defectsin mice lackingc-fos. Nature 360:741 -74S, 1992. 108. OukkaM. Ho IC. de la Brousse FC. HoeyT. GrusbyMJ. GlimcherLH: The transcription factorNFAT4 is involved in the generation and survival ofT-celis. Immunity 9:295-304. 1998. 109. SchwarzEM. BadorffC. HiuraTS. Wessely R, BadorffA. Verma 1M, Knowlton KU: NFkappaB-mediated inhibition of apoptosis is required for encephalomyocarditis virus virulence: a mechanism of resistance in pSO knockout mice. J Virol72:5654-5660. 1998. 110. Ishikawa H, ClaudioE. Dambach D. Raventos-Suarez C, Ryan C. BravoR: Chronic inflammation and susceptibility to bacterial infections in micelacking the polypeptide (p)IOS precursor(NF-kappaB I) but expressing pSO. J Exp Med 187:985-996. 1998. Ill. Carrasco D. ChengJ, Lewin A, WarrG, Yang H, RizzoC, Rosas F, SnapperC, BravoR: Multiple hemopoietic defectsand lymphoid hyperplasia in mice lacking the transcriptional activation domainof the c-Rel protein. 1 Exp Med 187:973-984, 1998. 112. ViolaIP. KianiA. BozzaPT.RaoA: Regulation of allergic inflammation and eosinophil recruitment in micelackingthe transcription factor NFATl : role of interleukin-4 (lL-4) and IL-S. Blood91:2223-2230.1998. 113. Franzoso G. Carlson L. PoljakL. ShoresEW. Epstein S, Leonardi A, Grinberg A, TranT. Scharton-Kersten T.AnverM. Love P,Brown K. Siebenlist U: Micedeficient in nuclear factor(NF)-kappa B/pS2 presentwithdefects in humoral responses, germinal centerreactions. and splenicmicroarchitecture. J Exp Med 187:147-159.1998. 114. Franzoso G, Carlson L, Xing L. PoljakL. ShoresEW. BrownKD. Leonardi A, TranT, BoyceBF.Siebenlist U: Requirement for NFkappaB in osteoclast and B-cell development. Genes Develll :3482-3496. 1997. lIS. Pruschy M. Shi YQ,Crompton NE.Steinbach J. AguzziA. Glanzmann C. BodisS: The proto-oncogene c-fos mediates apoptosis in murineT-Iymphocytes inducedby ionizing

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0, Li R, Tarlinton 0, Gerondakis S: Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression. Genes DeveI9:1965-1977,1995. 129. Beg AA, Sha WC, Bronson RT, Ghosh S, Baltimore 0: Embryonic lethality and liver degeneration in mice lacking the RelA component ofNF-kappa B. Nature 376: 167-170, 1995. 130. Sha WC, Liou HC, Tuomanen EI, Baltimore 0: Targeted disruption of the p50 subunit of NF-kappa B leads to multifocal defects in immune responses. Cell 80:321-330, 1995.

Assays for the Diagnosis of Recurrent Spontaneous Abortion of Immune Origin (Continuedjrompg.41)

some cases these can be diagnosed by chromosomal karyotyping. Other known causes of RSA are uterine abnormalities, microbial infections, or hormonal abnormalities," However, the majority of women (60 to 70%) with three or more spontaneous abortions have no demonstrable cause and are often considered to have immune-related RSA by default.!" The developing fetus is considered by some to be an allograft because as it develops, the molecules encoded by the father could be considered foreign by the mother and result in rejection. The primary organ of pregnancy is the placenta.l This organ is composed of multiple types of cells both embryo-derived and maternallyderived. The cells unique to the placenta are called trophoblasts. In fact, the placenta is encoded in large part by paternallyassociated and not maternally-associated genes so that the potential HLA exposure is primarily paternal and totally foreign to the maternal immune system. In most successful pregnancies, immune rejection of the fetus does not occur; however, strong evidence suggests that parturition itself may be in part immune mediated. In contrast, in RSA the fetus is rejected and pregnancy loss does result. Unless diagnosis and treatment of RSA occurs, this loss may occur multiple times. As stated initially, previously RSA of immunologic origin was thought to be a condition whose diagnosis was made after all other possibilities were eliminated.

131. Chen J, Stewart V, Spyrou G, Hilberg F, Wagner EF, Alt FW: Generation of normal T and B lymphocytes by c-jun deficient embryonic stem cells. Immunity 1:65-72, 1994.

135. Viola JP, Rao A: Role of the cyclosporinsensitive transcription factor NFATI in the allergic response. Memorias do Instituto Oswaldo Cruz 92: 147-155, 1997.

132. Baldwin AS, Jr.: The NF-kappa B and 1kappa B proteins: new discoveries and insights. Ann Rev ImmunoI14:649-683, 1996.

136. Liebermann DA, Gregory B, Hoffman B: AP· I (Fos/Jun) transcription factors In hematopoietic differentiation and apoptosis. Int J OncoI12:685-700, 1998.

133. Grigoriadis AB, Wang ZQ, Wagner EF: Fos and bone cell development: lessons from a nuclear oncogene. Trends in Genetics II :436441, 1995. 134. Karin M, Liu Z, Zandi E: AP-I function and regulation. CurrOpin Cell Bioi 9:240-246, 1997.

137. Attar RM, Caamano J, Carrasco D,lotsova V, Ishikawa H, Ryseck RP, Weih F, Bravo R: Genetic approaches to study ReVNF-kappa BII kappa B function in mice. Sem Can Bioi 8:93101, 1997.

There is now a greater understanding of the possible causes of RSA, and with this understanding comes the possibility of immunological testing. This can make the diagnosis of RSA of immune origin no longer a "rule-out" diagnosis. The laboratory tests that are used for the diagnosis of immune-related RSA are described below. When evaluating the immunologic characteristics of RSA, the unique immunologic environment of the placenta should be considered, The local milieu and events occurring at the maternal-fetal interface usually prevent an immune response against the fetus. The placenta acts as barrier protecting the fetus against maternal immune effector mechanisms and at the same time allows the passage of nutrients necessary for the developing fetus.' The trophoblasts in contact with maternal blood cells express the nonclassical HLA-G molecule rather than conventional class I and class II HLA antigens.v" While no clear data exists it is thought that HLA-G may prevent the initiation of an immune response and subsequent rejection. Some data suggests that attenuated cell-mediated immunity (CMI) and strong humoral immunity" characterize a normal, successful pregnancy. It has also been suggested that during pregnancy, down regulation of T helper type I (TH 1)associated cellular immunity may be crucial for maintenance of the fetal allograft.i" Some data suggests that there is a TH2 bias in normal pregnancy since mouse fetoplacental tissues secrete IL-4, IL-5, and IL-l 0. 8 It has been shown that less TH I cytokines and more TH2 cytokines

are produced during pregnancy in mice. THI cytokines (IL-2, IFN-y, and TNF-a and -6) may mediate several cytotoxic and inflammatory reactions which have been shown to be detrimental to pregnancy. These agents are known to damage the placenta directly or indirectly via activation of cytotoxic cells.' TH I cytokines activate natural killer (NK) cells and induce Iymphokine-activated killer (LAK) cells." Also, the increase in NK cells leads to increase in IFN_y.4 Trophoblast cells secrete cytokines that have been primarily associated with mononuclear phagocytes, such as CSF-I (and its receptor c-fms), IL-3, and GM-CFS. In contrast to normal pregnancy, several studies have indicated that women with a history of RSA demonstrate an abnormal pattern of lymphocyte distribution and activity.3,IO,1I During normal pregnancy, the levels ofNK cells in peripheral blood'? and their functional activity decreases.P In contrast, it has been shown that the numbers of NK cells do not decrease or may even increase in women with RSA. Indeed, peripheral blood CD56+ NK cells are significantly elevated in non-pregnant and pregnant women with a history of RSA as compared to pregnant controls. I I This phenomenon is perhaps due to damage to the placenta during pregnancy loss or proliferation of NK cells during abortion. I There may also be an elevation in circulating blood B-cells as compared to women with no history of RSA. II The unique nature of the lymphocytes have presented an opportunity for detecting immune-associated RSA. Pregnant women with a history of RSA

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0197·1859199 (see frontmatter)