Regulation and Function of Adenosine Deaminase in Mice

Regulation and Function of Adenosine Deaminase in Mice’ MICHAEL R. BLACKBURN AND RODNEY E. KELLEMS~ Verna and Marrs McLean Department of Biochemistry Baylor College of Medicine Houston, Texas 77030

I. Developmental and Tissue-specific Expression of Ada . . . . . . . . . . . . . . 11. Regulation of Ada Gene Expression . . . . . . . A. Gene and Promoter Structure . . . . . . . . . B. Tissue-specific Regulation ................................... C. Model for Ada Gene Expression .............................. .......... 111. Physiological Role of ADA during Development IV. Role of ADA in the Murine Immune System ...................... V. Role of ADA in the Secondary Deciduum . . . . . . . . . . . . . . . . . . . . . . . . . VI. Role of ADA in the Gastrointestinal Tract . . VII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ....................

198 203 203 204 207 208 216 219 220 221 223

Adenosine deaminase (ADA; EC 3.5.4.4) is an essential and widely distributed enzyme of purine catabolism that catalyzes the hydrolytic deamination of adenosine and deoxyadenosine to inosine and deoxyinosine ( 1 , 2 ) (Figs. 1and 2). The enzyme is present throughout the evolutionary phyla and the amino-acid sequence of the protein has been highly conserved from bacteria to humans (3). The catalytic moiety of ADA exists as a single polypeptide chain with approximately 83.1%amino-acid sequence identity between humans and mice (4,5). The human ADA open reading frame consists of 1089 nucleotides with a deduced sequence corresponding to a 40,762-Da peptide containing 363 amino acids (4, 6). The murine ADA open reading frame is 1056 nucleotides long and encodes a deduced 39,900-Da protein of 352 amino acids (5).Murine ADA lacks the 11 carboxy-terminal residues of 1 Abbreviations: ADA, adenosine deaminase; Ada, adenosine deaminase gene; 5’-NT, 5’nucleotidase; PNP, purine nucleoside phosphorylase; XO, xanthine oxidase; GDA, guanine deaminase; dpc, days postcoitum; CAT, chloramphenicol acetyltransferase; AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine. 2 To whom correspondence may he addressed.

Progress in Nucleic Acid Research and Molecular Biology, Vol. 55

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Copyright 0 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

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HOCHp

OH

OH

adenosine

inosine

0

OH

2deoxyadenosine

2-deoxyinosine

FIG. 1. Schematic of the ADA-catalyzed reaction showing the hydrolytic deamination of adenosine and deoxyadenosine to inosine and deoxyinosine.

the human protein; however, the importance of this region is not known. Recombinant murine ADA has been subjected to crystallographic analysis, and the architecture of the enzyme is a parallel a/p barrel with eight central (3 strands and eight peripheral a helixes (7). One zinc atom for each ADA molecule is tightly coordinated with three histidines, one aspartate, and a catalytic H,O molecule within the active-site pocket (8).Mutations that alter these amino acids abolish ADA enzymatic activity, suggesting that zinc is an essential cofwtor (9u). Functional studies indicate that ADAs from different species are similar with regard to substrate specificity and kinetic constants. The conservation of structure and function suggests that ADA plays an essential role throughout evolution. The importance of ADA for vertebrate systems stems primarily from the physiological impact of its substrates, adenosine and deoxyadenosine. Adenosine functions as an extracellular signal transducer that mediates a large

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1 J

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deoxyadenosine

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xanthinehric acid

FIG. 2. Purine catabolic pathway. 5’-Nucleotidase (5’-NT)catalyzes the dephosphorylation of adenosine 5’hionophosphate (5’-AMP) and deoxyadenosine 5’-monophospbate. Adenosine deaminase (ADA) catalyzes the hydrolytic deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, which are subsequently converted to hypoxanthine by purine nucleoside phosphorylase (PNP). Hypoxanthine can then be salvaged back to inosine monophosphate (IMP) by hypoxanthine phosphoribosyl transferase (HPRT), or can enter the nonsalvage pathway, where it is converted first to xanthine and then uric acid by xanthine oxidase (XO).

variety of physiological effects by binding to adenosine receptors present on the surface of target cells (10, 11). Deoxyadenosine behaves as a cytotoxic metabolite that can kill cells by interfering with deoxynucleotide metabolism and/or S-adenosylmethionine-dependent cellular transmethylation reactions (12).Deoxyadenosine cytotoxicity is believed to provide the metabolic basis for the immune deficiency that accompanies ADA deficiency in humans (13, 14). Because of the physiological importance of ADA, it is not surprising that the enzyme is ubiquitously distributed among vertebrate tissues (1, 15). In addition, the levels of ADA are developmentally regulated, and expression varies markedly among tissues (15-21), suggesting tissue-specific metabolic responsibilities for the enzyme. In mice, the tissue-specific differences in ADA enzymatic activity span a range of more than 10,OOO-fold (15). During prenatal development, the highest levels are present at the maternal-fetal interface throughout postimplantation stages of development (18-21). In the adult, the highest levels occur in the gastrointestinal tract (15).Within these tissues, the levels of ADA are

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subject to pronounced developmental regulation. Thus, the expression of the Ada gene is characterized by highly enhanced expression in a small collection of diverse tissues and a low level of expression in most other tissues. In this review, our current understanding of how this complex pattern of ADA expression is achieved is discussed. In addition, progress toward understanding the physiological importance of ADA in various tissues using transgenic mice, is reviewed.

1. Developmental and Tissue-specific Expression of Ada

A. Expression during Prenatal Development Very high levels of ADA are found at the maternal-fetal interface of mice throughout prenatal development in two genetically distinct tissues, the maternal deciduum and embryo-derived trophoblasts of the chorioallantoic placenta (18-21). ADA is initially of maternal origin and begins to accumulate in the gestation site 6.5 days postcoitum (dpc), 2 days after implantation. Immunohistochemical and in situ hybridization studies demonstrate that ADA synthesis at this time is primarily by secondary decidual cells (19, 20), which are specialized uterine stromal cells that surround the embryo on the antimesometrial half of the uterus (Fig. 3A). Examination of Ada expression during the development of experimentally induced deciduoma in rats and mice indicates that secondary decidual cells can synthesize high levels of ADA protein and mRNA in the absence of an embryo (22). Thus this initial phase of ADA synthesis at the maternal-fetal interface is almost exclusively maternal in origin. ADA expression is developmentally regulated and increases over 200fold to reach a maximum in secondary decidual cells by 9.5 dpc (Fig. 3A and C). Subsequently, this maternal source of ADA is lost to tissue regression, which is largely complete by 11.5 dpc (19). During this time the responsibility for synthesizing ADA shifts to embryo-derived trophoblast cells as they differentiate at the ectoplacental cone starting at 7.5 dpc, and in the giant cells that surround the implantation site. By 13.5 dpc, the junctional zone of the placenta is the major site of ADA synthesis in the gestation site. At this stage high levels of ADA are produced by all three trophoblast populations-labyrinthine trophoblasts, spongiotrophoblasts, and secondary giant trophoblasts-with the highest levels being found in the spongiotrophoblasts of the junctional zone (19,20) (Fig. 3B and C). This pattern of expression persists through term (23). Transfer of murine blastocysts carrying an electrophoretic variant of ADA into wild-type recipients was initially utilized

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FIG.3. Spatial and temporal pattern of ADA expression at the maternal-fetal interface. Paraffin cross-sections were reacted with a monospecific sheep antiserum to mouse ADA followed by immunoperoxidase detection (16).(A) By 9.5 dpc, ADA immunoreactivity is intense in the secondary deciduum, which by this stage is referred to as the decidua capsularis (dc). The mesometrial deciduum, also known as decidua basalis (db), is negative. Immunoreactivity is seen in trophoblasts of the labyrinthine (la) and junctional (basal) zone (bz) of the placenta. The embryo (located centrally) is negative. (B) By 13.5 dpc, the decidua capsularis has undergone massive regression (arrows), and ADA immunoreactivity is intense in the basal zone (bz) of the mature placenta. Reactivity is also seen in giant trophoblasts and the labyrinthine zone. The olfactory neuroepithelium (on) is indicated. (C)The temporal pattern of ADA enzymatic activity in the secondary deciduum and placenta is shown. Reproduced with permission from Knudsen et (11. (19).

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to demonstrate a major shift from maternal to embryo-derived ADA during midgestation (19). More recently, this has been shown by the use of ADAdeficient mice (Section V, B). Thus, the second phase of ADA synthesis at the maternal-fetal interface is provided predominantly by embryo-derived trophoblasts of the placenta. ADA enzymatic activity in the developing embryo and fetus is low relative to those found in the deciduum and placenta (19). However, immunoreactivity is detected in subsets of cell populations in the fetal central nervous system, including dorsal root ganglion (24)and the olfactory neuroepithelium (19). Activity has also been found in regions of the developing rat brain (25), and the developing gastrointestinal tract in the mouse also expresses ADA at birth (15).Taking into account total ADA enzymatic activity levels measured in the fetus and placenta, greater than 95% of ADA activity in the fetal gestation site is found in trophoblasts of the placenta (23).These high levels of ADA suggest there is an important role for ADA in purine metabolism during prenatal development in mice. Other enzymes of purine catabolism are found in the deciduum and placenta (20, 21) (Fig. 2), including 5’-nucleotidase (5’-NT),which produces ADA substrates from the dephosphorylation of AMP and dAMP (21).5’-NT is in the primary deciduum during implantation stages and in giant trophoblast cells of the developing and mature placenta. The levels of 5’-NT, however, are much lower than those measured for ADA. This pattern of 5’-NT expression suggests that there may be a need for adenosine synthesis during implantation and placentation. Indeed, endogenous adenosine levels surge in the pregnant uterus between 4.5 and 7.5 dpc (implantation stages) corresponding to elevations in 5’-NT enzymatic activity and low ADA enzymatic activity. Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO), enzymes of purine catabolism distal to ADA, are detected in the gestation site, but at low levels that do not change during development. Therefore, enzymes of the purine catabolic pathway do not appear to be coordinately expressed at the maternal-fetal interface in mice. ADA is by far the most expressed enzyme in the pathway, suggesting a physiological need for the control of ADA substrates during postimplantation development.

6. Postnatal Expression

Postnatally, the highest levels of ADA in the mouse occur in the gastrointestinal epithelium (Table I), where the enzyme accounts for more than 20% of the total soluble protein (15,26).ADA is in the keratinized squamous epithelium that lines the tongue, esophagus, and forestomach, and the simple columnar epithelium of the proximal small intestine (Fig. 4).In these tissues the level of ADA is subject to pronounced developmental control, being low at birth and achieving enormous levels within the first 5 weeks of postnatal

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TABLE I ADA ENZYMATIC ACTIVITY I N VARIOUSMOUSETISSUES~~ Tissue Gastrointestinal tract Tongue Esophagus Forestomach (nonglandular) Glandular stomach Duodenum Jejunum Ileum Large intestine Female reproductive tract Deciduum (experimentally induced) Embryo-deciduum unit (9,5 dpc)C Embryo (13.5 dpc) Placenta (13.5 dpc) Uterus Vagina Miscellaneous Thymus Spleen Liver Kidney Lung Bladder Ear pinme (skin) Foot pad (skin) Back (skin) Cardiac muscle Skeletal muscle ‘J

1,

ADA activity’)

5200 2200 4200 150 4200 1900 360 50 1400 1100 23 590 24 31 130 44 63 63 7 37 26 19 7 4 4

Data from Chiriskv et aZ. (15). Values are given as nmoliminimg protein

dpc, Days postroitum.

life (15) (Fig. 4G).Intermediate levels of ADA enzymatic activity are found in the thymus, spleen, and liver, whereas brain and heart are tissues that exhibit low levels of ADA enzymatic activity (15, 20) (Table I). In humans, as with the mouse, high levels of ADA are found in the proximal small intestine (20, 27). The human thymus also exhibits high levels of ADA enzymatic activity in cortical thymocytes (28). ADA is also expressed in the human deciduum and placenta, but at levels much lower than those measured in the mouse (29). Unlike the uterus in pregnancy, Ada expression in the gastrointestinal

0

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3 4 5 Age (weeks)

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FIG.4. ADA enzymatic activity in the postnatal gastrointestinal tract of mice. Immunofluorescent localization of ADA in the murine forestomach (a), esophagus (b), and proximal small intestine (c), using a monospecific antiserum to murine ADA. The arrows indicate the specific immunofluorescent staining in the mucosal epithelial layer (Mu); L, lumen; M,mucosal layer. ADA-formazan staining for catalytic activity is denoted by arrows in the forestomach (d) and small intestine (f). Ontogeny of ADA-specific activity in tissues from BALBlc mice at the postnatal ages indicated (9). Samples are given as means 2 standard deviations. Reproduced with permission from Dtfferentiation, Developmental expression of adenosine deaminase in the upper alimentary tract of mice, Chinsky et al., 42, 172-183, Figures 1 and 3, 1990, 0 SpringerVerlag.

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tract is accompanied by high levels of expression of other enzymes of the purine catabolic pathway (Fig. 2), including PNP, guanine deaminase (GDA), and XO (20, 30). The proximal small intestine of the adult is the richest source of these enzymes. Because they serve collectively to produce uric acid, it is possible that production of this antioxidant is important in gastrointestinal physiology. The diverse array of Ada expression during both prenatal and postnatal development suggests that this enzyme may serve different functions in different tissues, and demonstrates the complexity of Ada gene regulation. These issues are addressed below.

II. Regulation of Ada Gene Expression The mammalian genome consists of approximately three billion basepairs of DNA sequence information organized into an estimated 100,000 genes. The evidence suggests that approximately 25,000 genes are expressed in all tissues and that the remaining 75,000 genes are expressed in a tissuerestricted manner. The Ada gene has features of each category, i.e., ubiquitous expression and tissue-enhanced expression, making it an ideal model for studying developmental and tissue-specific gene regulation. Considerable progress toward defining key regulatory features of the human and murine Ada gene has been made.

A. Gene and Promoter Structure The complete nucleic acid sequence for the ADA gene in humans has been determined. The locus resides on chromosome 20 at q13.11 (31-33). The murine Ada gene is in the H2 region of chromosome 2 (34). The structures of the human and murine gene are similar (Fig. 5A and B), both containing 12 exons that span 32 kb (human) and 23 kb (murine) of nucleotide sequence. Intron 1 is large, spanning over 15 and 12 kb in the human and mouse, respectively (32, 35). Initial studies in the mouse determined that Ada promoter activity resides within a 240-bp fragment that contains the transcriptional initiation sites (36). Subsequent studies identified the position of core promoter and modulating elements within this region (37). Sequence analysis of this promoter region showed it to be extremely rich in G C (77%)and devoid of TATA and CAAT box consensus sequences (Fig. 5C) (37).This region contains multiple Spl binding sites, which bind purified Spl protein. The sequence TAAAAAA, found 27 bp upstream of the major transcriptional initiation site, together with at least one Spl site, is necessary for basal transcription in uitro. This A-rich sequence binds transcription factor IID (TFIID), which can promote transcription in uitro. Similar features have been shown for the human ADA promoter (38).

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MICHAEL R. BLACKBURN AND RODNEY E. KELLEMS

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FIG. 5. Structure of the human (A) and murine (B) A d a gene. Exons, denoted by black boxes, are numbered. Data for the human and murine gene are from Wiginton et al. (32)and AlUhaidi et al. (35),respectively. (C)Enlargement of the murine ADA promoter region. The arrow denotes the transcriptional start site and the black box represents exon 1. The TAAAAAA element is essential for transcription and is believed to bind basic transcriptional complexes. Other modulating elements are shown (37).

ADA enzymatic activity and the steady-state abundance of messenger RNA correlate in all tissues and cell lines examined. This suggests that

transcriptional regulation plays an important role in the expression of this gene. The 240-bp promoter region, alone or with the addition of 2.7 kb of 5’ flanking sequences, does not deliver enhanced expression of reporter genes in cell transfection studies or transgenic mice (36, 39, 40). Therefore, elements necessary for enhanced and tissue-specific expression reside elsewhere in the Ada gene.

B. Tiss u e- s pec ific Regu Iation 1.

REGULATION OF

PLACENTALEXPRESSION

Prenatally, A& is expressed at high levels in trophoblast cells of the developing and mature chorioallantoic placenta (19, 20). Ada expression appears in trophoblast cells as they differentiate from the ectoplacental cone, starting 7.5 dpc. In the mature placenta, Ada is highly expressed in the spongiotrophoblasts that comprise the junctional zone, in trophoblasts of the labyrinthine zone, and in giant trophoblasts that surround the implantation site. Studies in transgenic mice have been critical in capturing regulatory elements responsible for developmental and tissue-specific expression in the

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ADA REGULATION AND FUNCTION

placenta. A 6.4-kb fragment of DNA from the 5' flanking region of the murine Adu gene can direct high-level reporter gene expression to the placenta in a correct developmental manner, and to the forestomach postnatally, but not to any other tissue (23). Deletion analysis of this region revealed that placental and foresetomach enhancer sequences are separable (D. Shi, unpublished data). The placental enhancer was delineated to a 756bp fragment located between 5 and 6 kb upstream of the Ada transcription start site (Fig. 6). This fragment directed high levels of placental-specific chloramphenicol acetyltransferase (CAT) expression in transgenic mice. Furthermore, in situ hybridization studies monitoring the cellular localization of CAT transcripts showed that this placenta enhancer directed expression to all three trophoblast lineages at the appropriate levels. Insight into the mechanism by which the ADA placenta enhancer functions was provided by DNase-I footprinting assays and sequence analysis. DNase-I footprinting experiments revealed both general and placenta-specific protein-binding sites within this region (D. Shi, unpublished data). These regions may bind truns-acting factors necessary for placental enhancement. In addition, sequence analysis of this ADA placenta enhancer revealed that it contains sequence elements homologous to transcription-factor binding-sites found in other placental-specific genes. These include potential sites for the trophoblast-specific-element binding protein (TSEB) (42), zinc-finger transcription factors such as GATA-2 (43)and GATA-3 (44),CAMP 2

1

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FIG.6. Schematic of tissue-specific regulatory domains in the Ada gene. Depicted is a region of the Ada gene encompassing 5’ flanking sequences and the first three exons (black boxes) and introns. The locations of various regulatory doinains are shown. The bent arrow represents the transcriptional start site. This schematic is not to scale.

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MICHAEL R. BLACKBURN AND RODNEY E. KELLEMS

response-element binding proteins (45), and basic helix-loop-helix factors (46). This array of potential transcription-factor binding-sites is consistent with either the use of a single combination of sites capable of functioning in all three trophoblast cell types, or a unique combination of sites, one for each type. Studies are under way to test these possibilities by creating deletion and site-directed mutants of each site for expression in transgenic mice, using in situ hybridization to monitor transgene cellular localization. In addition, efforts to identify the proteins that bind these regulatory sequences are under way. It is possible that both novel and previously characterized trans-acting factors may interact with these elements. The identification of these factors will be an important step toward understanding Ada expression in trophoblast cells of the placenta, as well as defining the genetic regulatory pathways governing trophoblast differentiation.

2.

REGULATION OF

THYMUS EXPRESSION

DNA sequences within intron 1 of the human ADA gene confer highlevel expression of the CAT reporter gene to the thymus of transgenic mice and in permanent lines of human T cells (27, 47, 48). Analysis of DNase-I hypersensitive sites within intron 1 revealed a thymus-specific enhancer domain responsible for high-level thymus expression. The enhancer domain is 200 bp long and contains at least two critical core regions that bind ADANF1 (c-myb) and ADA-NF2 (47a). The enhancer also contains consensus matches for other potential transcription factors. Closer analysis revealed that sequences flanking the enhancer are required for positional independent and copy-number proportional expression of transgenes in the thymus of transgenic mice (Fig. 6), but not in permanent lines of human T cells (47, 48). These elements, which are required for enhancement in transgenic animals, are referred to as facilitators, and are believed to participate in establishing a chromatin configuration necessary for enhancer function (48). The comparable region of DNA from the murine Ada gene has been cloned and shows considerable regions of conservation with the human gene (J. H. Winston, unpublished data). A 3.6-kb intron-1 fragment encompassing this region was tested in transgenic mice using a CAT reporter gene. High-level CAT expression was seen in the thymus, suggesting that a thymus-specific enhancer for the murine Ada gene resides in a region of intron 1 similar to that of the human thymus enhancer (Fig. 6). Apart from increased expression in the thymus, the 3.6-kb fragment activated basal levels of CAT expression in all tissues examined, a feature also associated with the human Ada T-cell enhancer (27). Associated with this basal activation was the formation of a DNase-I hypersensitive site at the promoter of the transgenes, suggesting that sequences within intron 1 may promote basal activation by altering chromatin configuration at the promoter. It is speculated

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that sequences within intron 1 may function as a locus-control region ensuring that the ADA promoter is accessible to the basal transcription apparatus in all cells. It is not known if the facilitator sequences defined within intron 1 of the huhan gene are the same as the basal activator sequences found within intron 1in the mouse. Efforts to delineate these regulatory regions and determine what truns-acting factors are involved will contribute to our understanding of how the Ada gene is activated.

3.

REGULATION IN OTHER

TISSUES

Other tissues that exhibit high levels of Ada expression are tissues of the proximal gastrointestinal tract, including the squamous epithelium of the tongue, esophagus, and forestomach, and the absorptive columnar epithelium of the proximal small intestine (15). During reproduction, the secondary deciduum is highly enriched in ADA as well (19). Evidence suggests that, like the thymus and placenta, Ada expression in these tissues is regulated by specific enhancer sequences. DNA sequences in the 5’ flanking region, proximal to the placenta enhancer, are capable of directing expression of the CAT reporter gene to the forestomach of transgenic mice, but not to any other tissue (23). The forestomach enhancer lies within -2.7 to -5.0 kb relative to the Ada transcriptional start site (Fig. 6). From these findings, it is likely that regulatory sequences responsible for enhanced Ada expression in the tongue, esophagus, and proximal small intestine lie elsewhere within the gene. Current studies are under way analyzing regions of the murine Ada locus for such activities. Digestion of intact chromatin with DNase-I endonuclease is a means of identlfying regions of altered chromatin organization within a locus that may represent areas containing cis-regulatory elements involved in tissue-specific gene regulation. Analysis of DNase-I hypersensitive sites in the murine Ada gene revealed a strong decidual-specific hypersensitive site within intron 2 (L. Long, unpublished data) (Fig. 6). This region has a high degree of sequence homology with a similar region of intron 2 of the human Ada gene. This suggests a conserved function for this region of DNA, which may include enhanced expression of Ada in the secondary deciduum of the pregnant uterus. Studies are in progress to test functionally this region of D N A for its ability to drive expression of CAT reporter genes in a decidual-specific manner in transgenic mice. These transgenic approaches should allow us eventually to define functionally regions of the Ada gene necessary to accomplish its unique pattern of expression.

C. Model for Ada Gene Expression

The association between increased levels of ADA enzymatic activity and steady-state mRNA levels suggests that Ada expression is regulated at the

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level of transcription; however, the mechanisms involved are less clear. Nuclear run-on transcription experiments show an abundance of transcription complexes at the 5’ end of the Ada gene, in all tissues and cells tested. In contrast, probes representing downstream portions of the gene reveal high levels of transcription only in tissues and cells that are enriched for ADA (49-52). These findings suggest that the Ada promoter region is available to initiate transcription in all cell types, and that transcription continues only in those cells that are programmed for enhanced expression. Transcriptional studies in Xenopus laeois oocytes and in vitro have defined two transcriptional arrest sites in the 5’ end of the murine Ada gene, one within the 3‘ end of exon 1 and the other in the 5’ end of intron 1 (5357). It is not clear what role, if any, these sites might play in tissue-specific regulation of ADA. Additional studies show that the Ada promoter is readily accessible to DNase-I digestion in nuclei isolated from a variety of tissues, regardless of ADA levels (L. Hong, unpublished data). It is speculated that sequences within intron 1 may function as a locus control region, ensuring that the Ada promoter is accessible to the basal transcription apparatus in all cells (Section 11,B,2). Collectively, these data suggest that paused transcriptional complexes are likely present at the Ada promoter in all cells. The presence of 5’-paused transcriptional complexes has been proposed for other genes (58, 59), and the transcription of such genes is thought to be regulated b y modifications in the paused polymerase complexes that allow for transcript elongation. A potential model for Ada tissue-specific enhancement would involve the regulation of elongation of these paused transcriptional complexes, allowing them to elongate in tissues in which Ada is highly expressed. This could involve modifications in the paused complexes regulated by interactions with complexes associated with tissue-specific enhancer domains such as those defined for the thymus (27, 47, 48), placenta, and forestoinach (23).Continued efforts to define tissue-specific regulatory elements and the proteins that interact with them, and to test their interaction with transcriptional complexes, will be paramount to understanding the genetic mechanisms that regulate the complex pattern of Ada expression.

111. Physiological Role of ADA

during Development A. Genetic Disruption of Ada by Homologous Recombination

The use of homologous recombination techniques together with embryonic stem-cell manipulation has allowed for the targeted disruption of genes

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to assess their physiological function. A number of possible phenotypes can be predicted, based on the tissue distribution of ADA. The first tissue to

express ADA abundantly during development is the placenta (19, 20);therefore, an embryonic or fetal phenotype might be expected in the absence of placental ADA. During postnatal development, ADA is enriched in the proximal gastrointestinal tract (15), implying a possible gastrointestinal tract phenotype in ADA-deficient mice. By analogy to humans, the absence of ADA in the immune system, particularly the thymus, might result in lymphopenia or immunodeficiency. Finally, during reproduction, ADA is abundant in the secondary deciduum (19), suggesting a possible reproductive phenotype in female ADA-deficient mice. Thus, the generation of ADAdeficient mice would provide extensive information on the physiological importance of ADA in a variety of tissues, prenatally and postnatally. ADA-deficient mice have recently been generated by two independent groups, producing animals with independent sites of gene disruption (60, 61). In both cases, similar phenotypes were observed. Heterozygous matings produced Ada-null fetuses that died perinatally in association with severe hepatocellular impairment, incomplete expansion of the lungs, and small intestinal cell death. Liver damage was evident by 16.5 dpc and worsened through 18.5 dpc preceding death of the fetuses (60). This phenotype was accompanied by profound disturbances in purine metabolism. The lymphoid organs of ADA-deficient fetuses and newborn pups were not largely d e c t e d , although there were minor reductions in the number of CD4-positive and CDB-positive lymphoid cells in livers of ADA-deficient fetuses by 16.5 dpc (60).These observations suggest that ADA is essential for fetal survival in mice.

B. Metabolic Disturbances in ADA-deficient Mice In attempting to understand the metabolic basis for the liver damage and perinatal lethality seen in ADA-deficient fetuses, the levels of ADA substrates and products were assayed (60). Profound disturbances in purine metabolism were observed, with adenosine and deoxyadenosine increasing in ADA-deficient fetuses and levels of inosine decreasing (Fig. 7A). Substrates began to increase 12.5 dpc, and by 17.5 dpc adenosine and deoxyadenosine had increased 3-fold and 1000-fold (Fig. B), respectively. These observations suggest that disturbances in purine metabolism are likely involved in the mechanisms that lead to the phenotype observed in ADAdeficient fetuses. In attempting to understand the physiological importance of ADA, attention is invariably focused on the metabolic impact of its substrates, adenosine and deoxyadenosine (12). However, it cannot be ruled out at this point that the production of ADA-catalyzed reaction products may be necessary. However, it is difficult to envisage a need for inosine and/or deoxyinosine. The

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FIG. 7. HPLC chromatographic profiles showing purine metabolic disturbances in ADAdeficient fetuses. (A) Nucleoside profiles from wild-type (+/+) and homozygous mutant (-/-) fetuses 16.5 dpc. Peaks of interest include inosine (Ino), adenosine (Ado), and deoxyadenosine (dAdo). Notice that In0 is the major peak in the profile from the +I+ fetus, whereas Ado is low

ADA REGULATION AND FUNCTION

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days post coiturn

FIG.8. Schematic of phenotypes and temporal disturbances in purine nucleoside and nucleotide metabolism in ADA-deficient fetuses. Depicted are the phases of ADA expression at the maternal-fetal interface. Decidual expression is during early postimplantation stages and placental expression is during fetal stages of development. Slight increases in fetal ADA expression are also shown. The shaded box represents consequences observed in ADA-deficient fetuses, including the absence of placental and fetal ADA; the accumulation of adenosine, deoxyadenosine, and dATP; the appearance of liver damage 16.5 dpc; and perinatal lethality.

only known fate of each is to be phosphorylized to produce hypoxanthine and the respective sugar phosphate (Fig. 2). These compounds do not serve as precursors to critical metabolites that cannot be produced by other means. Thus, we do not believe it is likely that the liver pathology that accompanies ADA deficiency results from the inability of hepatocytes to produce inosine or deoxyinosine. Adenosine is an extracellular signaling molecule that elicits a vast array of physiological responses by engaging cell surface receptors (10, 11)(Fig. 9). Adenosine signaling is most often associated with cellular events that collectively serve to protect cells under metabolic stress, but can under some conditions be cytotoxic (62).Accumulations of adenosine seen in ADAdeficient fetuses (60) may disrupt adenosine signaling; however, little is known with regard to adenosine receptor types, levels, and localization in the murine embryo and fetus. Deoxyadenosine is a cytotoxic metabolite that kills target cells by interand dAdo is not detected. This pattern is reversed in -/- fetuses, in which Ino is reduced and Ado and dAdo are the major peaks found. (B) Nucleotide profile from whole blood collected from a heterozygous (+/-) and homozygous mutant (-/-) fetus 17.5 dpc. Whereas ATP is the major peak in blood from the +/- fetus, it is slightly reduced in blood from the - / - fetus. dATP levels are increased in -/- blood samples. The peak at 9.8 minutes in the -/- sample has tentatively been identified as dADP.

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FIG. 9. Schematic of pathways influenced by levels of adenosine and deoxyadenosine, which accumulate in the absence of ADA enzymatic activity. Adenosine (Ado)and deoxyadenosine (dAdo), generated by nucleic acid breakdown during apoptosis, are taken up by cells via a ubiquitously expressed nucleoside transporter. Extracellular (EC) Ado influences intracellular signaling by binding subsets of adenosine receptors (AR). Intracellular (IC) accumulations in dAdo can interfere with deoxynucleotide synthesis via its phosphorylation to dATP and suhsequent inhibition of ribonucleotide reductase. &do can also inhibit S-adenosylhomocysteine (AdoHcy) hydrolase, leading to disturbances in transmethylation reactions involving S-adenoxylmethionine (AdoMet). Accumulations of Ado can also influence this pathway by conversion to AdoHcy. Ino, inosine; dIno, deoxyinosine, NDPs, nucleotide diphosphates; dNDPs, deoxynucleotide diphosphates.

fering with deoxynucleotide metabolism and/or disrupting cellular transmethylation reactions (12) (Fig. 9). Interference with deoxynucleotide synthesis is mediated by the phosphorylation of deoxyadenosine to dATP via nucleoside and nucleotide kinases (63).Accumulation of dATP leads to the inhibition of ribonucleotide reductase and the disruption of deoxynucleotide synthesis needed for DNA replication and repair (13, 64, 65).Another route of deoxyadenosine cytotoxicity involves the inhibition of methylation reactions involving S-adenosylmethionine (AdoMet). The product of such methylation reactions is S-denosylhomocysteine (AdoHcy), which is hydrolyzed to adenosine and homocysteine by AdoHcy hydrolase. This enzyme is inhibited by deoxyadenosine, leading to the accumulation of AdoHcy, which can function as a competitive inhibitor of many transmethylation reactions critical to cellular function (66, 67). AdoHcy hydrolysis is reversible; therefore,

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accumulations of intracellular adenosine combining with free homocysteine can also cause increases in AdoHcy and subsequent inhibition of transmethylation reactions (67). These mechanisms of deoxyadenosine cytotoxicity are hypothesized to provide the metabolic basis for the immunodeficiency associated with ADA-deficient humans. In addition to marked accumulations in deoxyadenosine, concentrations of dATP are greatly elevated in the blood of ADA-deficient fetuses 17.5 dpc (60) (Fig. 7B). AdoHcy hydrolase is inhibited in livers and other tissues of ADA-deficient fetuses (M. Wakamiya, unpublished data) and pups (61), and this inhibition is accompanied by disturbances in the levels of AdoHcy and AdoMet, suggesting an interference in AdoMet-related cellular transmethylation reactions. These metabolic disturbances suggest that deoxyadenosine cytotoxicity (Fig. 9) is likely to provide the metabolic basis for liver damage and subsequent perinatal death of ADA-deficient fetuses. This hypothesis is further supported by the observation that the murine liver is a source of both deoxyadenosine-phosphorylatingenzymes and AdoHcy hydrolase (14, 68).

C. Genetic Reconstitution of ADA in the Placenta Over 95% of the ADA enzymatic activity in the fetal gestation site resides in trophoblasts of the placenta (19,23), suggesting an important role for ADA in this organ. The physiological importance of ADA in the placenta is not known; however, given that ADA-deficient fetuses, which also lack ADA in their adjoining placentas, die perinatally, it is reasonable to suggest that placental ADA plays an essential role during fetal stages of development. This hypothesis is supported by the observation that the metabolic disturbances seen in ADA-deficient fetuses are not evident until 12.5 dpc (60). This coincides with the removal of maternal ADA from the gestation site with regression of the secondary deciduum, and the failure of ADA to be expressed in ADA-deficient placentas (60) (Fig. 8). We assessed the importance of placental ADA by genetically restoring the enzyme to placentas of ADA-deficient fetuses (69). This was accomplished by designing an ADA minigene capable of targeting ADA expression to placentas of transgenic mice at levels comparable to that of endogenous ADA. This minigene was equipped with Ada placental gene regulatory elements (23)and modified to include a 36-bp deletion in the 5’ untranslated region, a feature that enabled us to distinguish the minigene transcript from native ADA transcripts. Once transgenic mice carrying this minigene were generated, they were intercrossed with mice heterozygous for the null Ada allele. Among the progeny of such matings were animals hemizygous for the ADA minigene locus and heterozygous for the null Ada allele. When intercrossed, these animals served as a source of fetuses that were homozygous for the null Ada allele, some of which carried the ADA minigene locus.

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Consistent with previous results (60),no ADA-deficient mice were present at weaning; however, ADA-deficient mice carrying the ADA minigene locus were detected at a percentage suggesting a 100% rescue of these animals (69). Thus, the expression of the ADA minigene in the placentas of ADA-deficient fetuses was sufficient to rescue them from perinatal lethality, suggesting that placental ADA may play an important role during fetal stages of development.

D. Prevention of Metabolic Disturbances by Placental ADA Expression On closer examination of rescued fetuses, it was found that severe fetal liver damage was prevented. In addition, most of the purine metabolic disturbances found in ADA-deficient fetuses (60) were prevented by the expression of the ADA minigene in their adjoining placentas (Fig. 10). This included the prevention of deoxyadenosine accumulation in ADA-deficient fetuses and placentas (Fig. 10B and E), and the prevention of dATP accumulation in fetal blood (Fig. 1OC). These findings further suggest that the high levels of ADA found in the murine placenta are important for fetal development, and a major function of this enzyme in the placenta is likely to prevent the accumulation of deoxyadenosine, which is potentially toxic to the developing embryo and fetus. Interestingly, the accumulation of adenosine was not prevented in ADAdeficient fetuses expressing an ADA minigene in their placentas, but was prevented from accumulating in the placenta itself(Fig. 10A and D). Little is known with regard to the involvement of adenosine signaling during mammalian development. The failure to prevent adenosine accumulation in rescued ADA-deficient fetuses suggests that elevated adenosine levels are not overtly detrimental to these fetuses. However, the potential effects of high concentrations of adenosine on placental development and function are not clear. Adenosine receptors are known to be expressed in the murine placenta (70).The prevention of adenosine accumulation in the placentas of rescued fetuses raises the possibility that high levels of adenosine in the placenta may exert a detrimental effect on placental function in ADA deficient mice by interaction with receptors. However, ADA-deficient and rescued placentas appear morphologically normal. More knowledge regarding the expression of adenosine receptors in the placenta and fetus are needed before we can assign a role to adenosine signaling in the phenotypes observed. Naturally occurring null alleles of the Ada gene have been observed in the human population and are associated with ADA-deficient immunodeficiency (12).In contrast to what we observed in mice, human ADA-deficient fetuses survive prenatal development and are not known to suffer severe hepatocellular damage, although mild liver findings have been noted (12). It is possible that fundamental differences in how human and murine hepato-

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FIG.10. Levels of ADA substrates and dATP in fetuses and placentas 17.5 dpc. Fetuses and placentas from intercrosses between mice heterozygous for the null Ada allele and hemizygous for the ADA minigene locus (Tg) were collected 17.5 dpc, and nucleosides and nucleotides were extracted and analyzed using reversed-phase HPLC. (A) Fetal adenosine levels. (B) Fetal deoxyadenosine levels. (C) dATP levels measured in fetal blood. (D) Placental adenosine levels. (E) Placental deoxyadenosine levels. Measurements were made on samples heterozygous for the null Ada allele with the ADA minigene locus (Tg, m l / + , n = 4), and samples homozygous for the null Ada allele without (ml/ml, n = 2) or with (Tg, ml/ml, n = 2) the ADA minigene locus. Tg,m l / + values are essentially the same as wild-type values. Values are given as means 2 SE; N.D., not detected at a lower limit of detection of 50.001 nmolimg protein. Reproduced by permission from Blackburn et al. (69).

cytes respond to alterations in ADA substrates may be responsible for the d8erence in phenotypes. Alternatively, these differences may suggest that there is a greater need for ADA during mouse prenatal development. This

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need may stem from a higher rate of ADA substrate generation resulting from tissue degeneration, which occurs at the maternal-fetal interface throughout murine development as a natural part of implantation chamber reorganization (71, 72). Genetic restoration of ADA in the placentas of ADAdeficient fetuses allows them to survive prenatal development, providing the opportunity to investigate whether postnatal mice develop phenotypes similar to those seen in humans (see Section IV).

E. Tissue-specific Rescues: A General Strategy The rescue of ADA-deficient fetuses by genetically restoring ADA to the placenta illustrates a general strategy for the tissue-specific correction of phenotypes associated with null mutations in mice. It is not uncommon for null mutations generated by gene targeting to result in embryonic or fetal lethality (73). Whereas this demonstrates the importance of a gene during prenatal development, it prevents investigation of its function postnatally. For example, ADA is expressed at high levels at three different places and times during the murine life cycle: first in the trophoblasts of the placenta during prenatal development (19,2O); next in the gastrointestinal epithelium of the adult (15);and then in the secondary deciduum of the pregnant uterus (19). The lack of ADA in the first of these places, the placenta, results in a phenotype that precludes our ability to investigate its importance in adult tissues. One approach to such problems is to create a tissue-specific knockout of a gene by strategies that employ the recombinase Cre (74).An alternative approach was demonstrated here, and involved tissue-specific restoration of gene expression on a knock-out background. Placental expression of an ADA minigene on an ADA-deficient background allowed for the survival of ADA-deficient fetuses through the prenatal phenotypic bottleneck, and provided adult mice that were used to assess the role of ADA in adult tissues, such as the gastrointestinal tract, the deciduum, and the immune system. Similar approaches may be useful in assessing the physiological role of genes in animals carrying targeted mutations that result in placental-related embryolethalities that prevent postnatal evaluations of these genes (75-77).

IV. Role of ADA in the Murine Immune System A. Lymphopenia and Immunodeficiency Associated with Restricted Expression of ADA Depending on the severity of the mutation, ADA deficiency in humans is associated with mild to severe immunodeficiency that, in severe cases, can lead to infant mortality, if not treated (12).The DNA fragment used to target

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expression of the ADA minigene in transgenic mice contains regulatory elements for high levels of expression in the placenta prenatally and the forestomach postnatally (23).Consistent with this, ADA enzymatic activity in rescued mice is only in the forestomach and to a lesser extent in other regions of the gastrointestinal tract (78). On investigating the status of the immune system in these rescued mice with restricted Ada expression, the spleen and thymus were found to be smaller and lymphoid cell counts were significantly reduced in these major lymphoid organs (Fig. 11). This suggests that these mice exhibited lymphopenia. Further evaluation by flow cytometry of leukocyte subpopulations in

C

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FIG. 11. Reduction in lymphoid organ size and cell number in adult mice with restricted ADA expression. Adult mice, rescued by the expression of the ADA minigene locus (Tg) in the placenta prenatally, did not express ADA in any tissue outside the gastrointestinal tract. Spleens (A) and thymuses (B) from 12-week-oldTg mice heterozygous for the null Ada allele (Tg, ml/+) and Tg mice homozygous for the null Ada allele (Tg, m l / m l ) , showing the reduction in lyniphoid organ size in the absence of ADA. Similar reduction in organ size has been observed in all animals examined thus far. (C) Total lymphoid cells counted in spleens and thymuses from Tg, m l i f and Tg, inUmI literrnates ranging from 9 to 13weeks of age. Two females and two males were examined for each genotype. Data (78)are given as total lymphoid cells (in millions) SD; *, significant with P < 0.02.

*

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spleens and thymuses from these mice showed that there were no significant differences in the distribution of leukocyte subpopulations in the thymus. However, leukocyte subpopulations were significantly altered in the spleens. The most striking change was a decrease in spleen T-cell populations, with a significant reduction in CDPpositive cells. A slight reduction in CD8-positive T cells and moderate increases in B cells (CD45R positive) were also observed. Consistent with this lymphopenia, lymphoid cells from rescued mice showed reduced responsiveness to mitogen stimulation, suggesting a partial immunodeficiency. In humans, ADA deficiency is associated with a mild to severe immunodeficiency, depending on the severity of the mutation in the Ada gene. T-cell lymphopenia often precedes overt immune deficiency in patients with milder mutations (79), and may account for some cases of “HIV-negative CD4 lymphocytopenia” (12). Mice with restricted Ada expression resemble these patients in that they have lymphopenia and partial immunodeficiency (78). Similar results have been observed in mice chronically treated with the ADA inhibitor 2’-deoxycoformycin (SO). The usefulness of rescued partially ADA-deficient animals as models for immune development and diseases awaits more thorough characterization of their immune system. However, it appears that these mice will be useful as animal models for studying the metabolic basis of ADA-related immunodeficiencies in humans (Section IV,B). In addition, these animals will be useful as tools for the advancement of enzyme and gene therapy approaches to treating ADA deficiency in humans. Current efforts are under way to rescue ADA-deficient fetuses using placental-specific DNA elements, which are expected to yield postnatal animals completely deficient in this enzyme. It is speculated that these animals will exhibit more severe lymphopenias and immunodeficiencies.

B. Metabolic Basis for Partial Immunodeficiency Purine metabolic disturbances are readily detected in patients with partial and complete ADA deficiency (65, 79, 81). These disturbances include elevations in plasma adenosine and deoxyadenosine, deoxyadenosine in the urine, elevated dATP and decreased ATP in erythrocytes, and decreased AdoHcy hydrolase activity in erythrocytes. Deoxyadenosine cytotoxicity is thought to provide the metabolic basis for the immunodeficiencyobserved in ADA-deficient humans (65); however, adenosine has also been shown to be toxic to T lymphocytes (62). Severe disturbances in purine metabolism have been observed in rescued mice with restricted Ada expression (78). Adenosine levels were elevated in the thymus, spleen, liver, kidney, and serum, whereas pronounced elevations of deoxyadenosine and dATP were observed only in the thymus of rescued animals. There was also an inhibition of

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AdoHcy hydrolase in the spleen, thymus, and liver of rescued mice examined. Similar metabolic disturbances have been reported in mice treated with the ADA inhibitor 2’-deoxycoformycin (82). These findings strongly suggest that mice with limited Ada expression suffer lymphopenia and partial immunodeficiency resulting from deoxyadenosine cytotoxicity.

V. Role of ADA in the Secondary Deciduum A. Pharmacologic Inhibition during Early PostimpIa ntat ion Stages During early postimplantation stages of development, ADA is abundant in the secondary deciduum surrounding the embryo (Fig. 3A and C) (19).As with the high levels of ADA in the placenta, high-level decidual ADA expression suggests that it may serve an important role during early postimplantation stages of development. Treatment of pregnant mice with the potent ADA inhibitor 2’-deoxycoformycin, 7.5 and 8.5 dpc, resulted in a high incidence of embryo lethality, which was evident by 12.5 dpc (83, 84). The teratogenic effects of 2’-deoxycoformycin coincide with these stages when decidual ADA is elevated. Inhibition of ADA in the gestation site was accompanied by disturbances in purine metabolism, including local increases in adenosine and deoxyadenosine (85), as well as dATP (86).Massive apoptosis was seen in embryos shortly after 2‘-deoxycoformycin exposure, and in vitro studies suggest that the accumulation of deoxyadenosine was responsible for the induction of apoptosis (86).The phenotypic and metabolic effects seen were dose dependent, stage specific (7.5and 8.5 dpc only), and stereoselective for the inhibitor, and suggest that ADA may be playing an important role during early postimplantation stages of development.

B. Reproductive Status of ADA-deficient Mice Pharmacological studies with 2’-deoxycoformycin have involved the administration of the inhibitor to the whole animal, making it difficult to determine the relative roles of placental, embryonic, or decidual ADA during early postimplantation stages of development. The creation of ADA-deficient animals, followed by the correction of the placental phenotype, produced mice lacking ADA in all tissues outside the gastrointestinal tract, including the secondary deciduum. These mice provided an opportunity to test genetically the importance of decidual ADA. Intercrosses between rescued male and female mice with restricted Ada expression were reproductively successful (M . R. Blackburn, unpublished data). The females used in these studies lacked ADA in their deciduum.

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However, because of the presence of the ADA minigene locus, they expressed normal levels of ADA in trophoblasts of their placentas. Subsequent matings between females lacking decidual ADA and males heterozygous for the null Ada allele, but lacking the ADA minigene, produced litters consisting of gestation sites, all of which were devoid of decidual ADA, and some which did or did not contain placental ADA. These litters were smaller than those of homozygous mating pairs; histological and iininunohistochemical analyses revealed that gestation sites lacking both decidual and placental ADA showed signs of embryolethality by 9.5 dpc. These data suggest that ADA at the maternal-fetal interface during early postimplantation stages is essential for normal development. However, the relative importance of decidual or placental ADA is still unclear. Though the physiologic mechanisms involved are still not known, it appears that ADA provided by the ADA minigene locus in the developing placenta is sufficient to allow ADA-deficient embryos to survive early postimplantation stages of development without decidual ADA. Massive cell death occurs at the maternal-fetal interface in the mouse as a natural part of the implantation process and the outward growth and expansion of the embryo and placenta (71, 72). Similarly, apoptosis is part of the developmental process of most organ systems in the developing embryo and fetus. Deoxyadenosine is generated from the breakdown of DNA from dying cells (12).The sensitivity of the embryo and fetus to deoxyadenosine cytotoxicity (60, 86) suggests that there must be a means to protect the embryo and fetus from deoxyadenosine accumulation. It seems likely that the high levels of ADA at the maternal-fetal interface serve to prevent the accumulation of deoxyadenosine generated by local cell death. Assessment of the metabolic changes that occur in the embryo and fetus in the presence or absence of decidual and/or placental ADA will strengthen this hypothesis.

VI. Role of ADA in the Gastrointestinal Tract The highest levels of Ada expression in the adult mouse are found in the proximal gastrointestinal tract (Table I) (15,30). Immunohistochemical analysis revealed the enzyme to be predominantly localized to the keratinized squamous epithelium of the tongue, esophagus, and forestomach, and the simple columnar epithelium lining the villus of the small intestine (Fig. 4) (15).The physiological role of ADA in these tissues is not known; however, ADA is part of a collection of purine catabolic enzymes that are highly expressed in the proximal small intestine and that function to produce uric acid (30,27)(Fig. 2). One possible function of ADA in the small intestine is to participate in the production of this antioxidant, which may be important in

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trapping peroxyl radicals formed during digestion (30). As the simple columnar epithelial cells of the small intestine differentiate and migrate to the tips of the intestinal villi, they undergo apoptosis and are then shed into the lumen. This cell death is a potential source of deoxyadenosine, which may be harmful to local tissues such as the mucosally associated lymphoid tissue (87). High levels of ADA in these cells may be present to prevent deoxyadenosine from accumulating. This is consistent with a potential role of ADA in other tissues that undergo massive apoptosis, such as the thymus (12, 28) and deciduum (71, 72). However, ADA is not found in all tissues that exhibit apoptosis. Excessive apoptosis has been observed in the proximal small intestine of newborn ADA-deficient fetuses (61).This suggests that ADA may be playing an important role in this tissue. However, the perinatal lethality of these mice due to liver damage has thus far prevented the full manifestation and characterization of this phenotype. Gross examination of ADA-deficient mice rescued by placental ADA restoration suggests that there are no severe structural problems in the gastrointestinal tract of mice that express Ada only in their forestomachs. It is possible that the lack of normal amounts of ADA in the gastrointestinal tract may cause disturbances in uric acid production, which may effect gastrointestinal physiology. More information regarding the physiological importance of ADA in the proximal gastrointestinal tract awaits the outcome of ongoing efforts to generate rescued animals that are completely ADA deficient. Expression of ADA in various regions of the gastrointestinal tract appears to be regulated by separate tissue-specific gene regulatory elements (Section II,B,3) (23).As with placental Ada regulatory elements, these gastrointestinal tract elements will be useful for targeting expression of regulatory molecules to various regions of the proximal gastrointestinal tract to assess their physiological roles.

VII. Summary Much has been learned about the human and mouse Ada genes: they are similar in their primary structure, they appear to be regulated in similar fashions, and their gene products play similar and critical roles in controlling the levels of physiologically active purines. ADA is an essential enzyme of purine metabolism, which is ubiquitously expressed, but also displays enhanced expression in specific tissues during development and in the adult. In mice, Ada is highly expressed prenatally at the maternal-fetal interface, first in the maternal deciduum during early postimplantation stages, and then in embryo-derived trophoblasts of the placenta during fetal stages of development (18-21). Postnatally, ADA is found at high levels throughout

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the proximal gastrointestinal tract (15, 26). There is still much to be learned with regard to how this pattern of expression is achieved; however, there is

mounting evidence that enhanced expression is regulated by distinct gene regulatory elements located throughout the Ada gene. Transgenic mice have proved to be critical for deciphering these regulatory elements, due to the complexity of Ada expression patterns and the need to monitor expression during the normal developmental program (23). The most thoroughly characterized enhancer domain is that required for enhanced expression in the human thymus. Both humans and mice contain a conserved enhancer domain within intron 1, which is necessary for enhanced expression in the thymus (27, 47, 48). A unique feature of this domain in the human is that it requires sequences referred to as “facilitators,” which establish a chromatin transition state necessary for the T-cell enhancer domain to function (48). Similar regions of the murine Ada gene possess the ability to activate the Ada promoter in all tissues. Considerable progress has been made in the identification and characterization of a placental enhancer in the mouse. It is located within the 5’ flanking region of the Ada gene and possesses placental nuclear-protein binding sites as well as many potential transcription-factor binding sites that may be important in placental-specific expression of ADA (23, 41). Other gene regulatory elements that are in the process of being characterized include forestomach, decidual, and small intestine tissue-specific enhancers. Deciphering gene regulatory elements responsible for the developmental and tissue-specific expression of Ada will improve our knowledge of how this gene accomplishes its unique pattern of expression, as well as increase our understanding of gene expression in general. In addition, defining tissue-specific gene regulatory elements will enable the targeting of selected cDNAs to the various tissues in transgenic mice. This will provide a new and powerful means of addressing the functional roles of various regulatory molecules within these tissues. For example, the identification of regulatory molecules and their receptors in subsets of trophoblasts at various stages of development has given rise to new hypotheses regarding mechanisms of implantation and placentation. These molecules include cytokines and their receptors, and matrix-degrading enzymes and inhibitors of these enzymes. Placental regulatory elements can now be utilized to miss-express these proteins, or mutant forms of them, to assess their physiological roles in the placenta. Another example of how these elements can be utilized is seen in the rescue of ADA-deficient mice through the restoration of placental ADA (69). This illustrates a general strategy for the tissue-specific correction of phenotypes associated with null mutations in mice. Placental ADA accounts for

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over 95% of the ADA found in the fetal gestation site (23). ADA-deficient fetuses that also lack ADA in their adjoining placentas die perinatally in association with profound purine metabolic disturbances and hepatocellular impairment (60, 61). The importance of placental ADA was shown by genetically restoring the enzyme to the placentas of ADA-deficient fetuses (69). Doing so prevented most of the purine metabolic disturbances as well as severe liver damage. This suggests that disturbances in purine metabolism, particularly those related to deoxyadenosine cytotoxicity, are responsible for liver damage and subsequent perinatal lethality. The resulting postnatal animals have restricted ADA expression and show signs of CD4 lymphopenia and immunodeficiency, which were associated with thymus-specific accumulations in deoxyadenosine and dATP and profound disturbances in AdoHcy hydrolase metabolism (78). This genetic strategy has now provided animals that can be used to address long-standing questions with regard to the metabolic basis for the immunodeficiency associated with ADA-deficient humans. These animals can also be utilized in the development of enzyme and gene-therapy approaches to treating immunodeficiencies as well as developing new strategies for treating leukemias susceptible to disturbances in purine metabolism. It will be of interest to decipher the mechanism of liver toxicity in ADAdeficient animals, and to assess why this phenotype is not seen in humans lacking ADA. Finally, continued examination into the role ofADA at the maternal-fetal interface should provide compelling information into the control of physiological purines during development. In conclusion, the diverse pattern of Ada expression provides an excellent model for studying gene regulation, as well as tissue-specific responsibilities of this enzyme. ACKNOWLEDGMENTS We thank John Winston, Vera Sidaraki and David Wilson for their critical review of this manuscript. This work was supported by NIH Grants GM42436, DK46207, and HD30302. M. R. B. was supported by a NIH postdoctoral fellowship (HD07843).

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