[Advances in Enzymology - and Related Areas of Molecular Biology] Advances in Enzymology and Related...

EXPRESSION OF THE CX-CRYSTALLINI SMALL HEAT-SHOCK PROTEIN/ MOLECULAR CHAPERONE GENES IN THE LENS AND OTHER TISSUES

B y C H R I S T I N A M . S A X and J O R A M P I A T I G O R S K Y , L a b o r a t o r y of Mole L' u l a r a n d D e v e I o p in e n t a I B i o 1 o g y , N a t i o n a l E y e I n s t i t u t e , N u t i o n a l I n s t i t r i t e s o.f H e a l t h , B e t h e s d a . M D

C O N T E N T S

I . Introduction 11. The a-CrystallinsisHSPs

A. B. C.

Ancestral Relationship of a-Cryskillin to sHSPs Structure and Evolution of the a-CrystallinisHSP Genes NonLens Expression of the a-Crystallins I . aB-Crystallin 2. aA-Crystallin

Expression of aB-Crystallin in Disease D. Experimental Induction of aB-Crystallin E. F. a-CrystallinsisHSPs as Molecular Chaperones G. a-Crystallin has Autokinase Activity Regulation of a-Crystallin Gene Expression A. aA-Crystallin

111.

I . 2. Mouse aA-Crystallin Gene Regulation 3. Chicken aA-Crystallin Gene Regulation

I . Comparisons of 5' Flanking Sequences 2. Transgenic Mice 3. The aB-Crystallin Enhancer 4. Lens Expression 5 . Alternative Transcription Initiation Sites 6. Stress Induction

Partial Sequence Conservation and Functional Divergence

B. aB-Crystallin

IV. Conclusions Acknowledgments References

I55

Advances in Enzymology and Related Areas ofbfolecular Biology, Volume 69 Edited by Alion Meisier

Copyrighi © 1994 by John Wiley & Sons, Inc.

156 CHRISTINA M . SAX AND JORAM PIATIGORSKY

I. Introduction

Vision is a complicated process requiring the coordination of environmental light, numerous eye tissues, and brain function. The transparent cornea and lens refract light rays onto the retinal photorecep- tor cells, which transform photons into nerve impulses and transmit them to the brain for interpretation. In humans, the ability to focus is gradually lost with aging, impairing vision ( I ) . The gross structure and function of the eye lens have been highly conserved in vertebrates (2). In addition, lenses in vertebrates and some invertebrates have acquired remarkable similarities by convergent evolution (3, 4). Accumulation of high concentrations of certain soluble proteins-called crystallins-is a characteristic feature of the transparent lens and is important for its optical functions ( 5 , 6, 7). There is a smooth gradient of refractive index from the center to the periphery of the lens resulting from the concentration of crystallins, which display short-range interactions (8, 9).

The mature avascular lens is a cellular, noninnervated tissue composed of anterior cuboidal epithelial cells and posterior elongated fiber cells contained within a capsule ( I , 10, I I ) . Of special importance is the fact that the cell nuclei are lost by pyknosis in the central fiber cells, making it impossible for the crystallins to turn over and requiring them to withstand a lifetime of environmental insults. A diagram of a human lens is shown in Figure 1.

There is a surprising diversity of crystallins despite their specialized function for lens refraction (7, 12, 13). While the a- and py-

Central Epithelial Cells A ,

Capsule

Fiber Cells Fiber Cells

Figure 1. Diagram of the mature human lens (from ref. I ) .

a-CRYSTALLIN GENE EXPRESSION 157

crystallins are present in all vertebrate lenses, another group of taxon-specific crystallins are present only in specific phylogenetic groups. These are either related or identical to metabolic enzymes and consequently are called enzyme-crystallins (14, 15). Although the basis for selection of individual crystallins is not known, it is intriguing that many are derived from metabolic enzymes or active stress proteins that can protect cells against environmental insults (such as changes in temperature, osmotic pressure, or oxidation state) or aging (16). We have called the dual use of an active enzyme or stress protein for a metabolic role and refractive function gene sharing (14, 17). The use of gene sharing for recruitment of lens crystallins has indicated that a protein can acquire a new function (in this case refraction) without gene duplication or without losing its original function by the modification of the expression of its gene (18).

The a-crystallins are a particularly interesting example of gene sharing since they are among the predominant crystallins of vertebrate lenses and are members of the small heat-shock proteins (sHSPs) (19). Here we summarize current studies on a-crystallin gene expression, with particular attention given to molecular investi- gations. Much of this work is being performed currently, and the ideas are changing rapidly. Thus, the purpose of the present review is to organize and evaluate the state of the art at the time of writing.

11. The a-Crystallins/sHSPs

A. ANCESTRAL RELATIONSHIP OF a-CRYSTALLIN TO sHSPS

The discovery that the a-crystallins are homologous to the sHSPs of Drosophilu was the first indication that the lens crystallins have been derived from ubiquitously expressed proteins with nonrefrac- tive functions (20). The wealth of sequence data that has accumu- lated has established that the a-crystallins are members of a superfamily of sHSPs. This sHSP superfamily also includes a major egg antigen from Schistosoma mansoni, several mycobacterial surface proteins, and numerous plant proteins, including a chloroplast-located class (19). As expected, many of the properties of the a-crystallins are shared with the known sHSPs (Table I ) . Indeed, the a- crystallins and sHSPs have been shown to copurify and interact with

I58 CHRISTINA M. S A X A N D JORAM PIATIGORSKY

TABLE I Properties of a-Crystallin and sHSPs

~

- have 20 kd subunits with p-sheet structure - form 800 kd aggregates - are thermostable - confer thermostability to cells" - are phosphorylated at specific serines - are glycosylated with 0-GlcNac linkage - possess chaperone activity - possess protease inhibitory activity - have possible tetramer-based quaternary structure ( 157) - increase aggregation during aging and heat-shock - relocalize from soluble cytoplasmic fraction to crude nuclear fraction under heat

- can donate amines for transglutaminase activity" - can associate with the cytoskeleton" - possess kinase activity (84d)

shock"

I' Properties that have not yet been established for aA-crystallin.

Reviewed in 5 and 19, except where additional reference is given.

one another (21, 22, 23). Recent sequence comparisons have also suggested more distant similarities between functional domains of sHSPs and the large heat-shock protein, HSP70, of eukaryotes and prokaryotes (24).

B. STRUCTURE AND EVOLUTION OF THE a-CRYSTALLINkHSP GENES

There are two highly conserved a-crystallin genes (aA and aB) in all vertebrate species examined (Figure 2). The 20-kD aA- and B- crystallin proteins are approximately 60% identical in amino acid sequence; it has been estimated that these two related proteins arose by gene duplication as long as 750 million years ago (2). In general, the a A and a B genes are located on different chromosomes (7); in humans the aA-crystallin gene maps to chromosome 21 (25) and the aB gene to chromosome I I (26, 27). The aA- (28, 29, 30, 31) and aB-crystallins (32, 33) are encoded by three exons in all species examined. Rodents (34, 35) and some other mammals (36) have an additional aA-crystallin polypeptide called &Ains (Figure 2). This variant polypeptide arises as a result of the alternative RNA splicing of an exon (the insert exon) within the first intron; in the mouse (28) and hamster (29), the insert exon encodes 23 amino acids. In mice

a-CRYSTALLIN GENE EXPRESSION I59

sHSP25/27 LENS Colon, ovary, uterus, heart, lung, brain Stressinducible

1 ins 2 3

d-crystallin a64rystallin LENS LENS Spleen, thymus Trace amounts in other tissues

Heart, skeletal &smooth muscle, kidney, lung, central &peripheral nervous svstem, retina, iris, thvroid. colon, s&amous epithelium, placenta, sper- matocyte Stress-inducible

Figure 2. a-Crystallin and sHSP gene structures. Schematic diagrams of the aA- (28. 29, 30, 31) and cuB-crystallin (32. 33) and sHSP25127 genes. sHSP2S (39, 40) is the mouse gene and sHSP27 (41) is the human gene. Open, numbered boxes represent exons present in all species examined, and the stippled box represents the insert exon (ins) (28, 29). The tissues in which each gene is expressed are also listed. LENS is emphasized because it contains the highest concentration of these proteins.

approximately 20% of the aA-crystallin mRNAs contain sequences derived from the insert exon (37). There is no evidence that the alternative RNA splicing of the aA-crystallin gene is a developmentally controlled event (37). The human gene contains a remnant of the insert exon that is not used to generate mRNA; it has accumu- lated numerous mutations and is considered a pseudo-exon (38).

In addition to the sequence similarities of the sHSPs and the ci- crystallins, the exon-intron structures of the mouse sHSP25 (39.40) and human sHSP27 (41) genes appear similar to those of the aA- and aB-crystallin genes (Figure 2 ) . Moreover, the a-crystallin and the sHSP25 genes function constitutively most highly in the lens and

160 CHRISTINA M. SAX AND JORAM PlATlGORSKY

to a lesser extent in numerous other tissues, although it is important to underscore that the expression patterns of the genes are not identical (42) (Figure 2). A particularly interesting difference between the aA- and aB-crystallin genes is that only the latter has been shown to be stress-inducible as an sHSP gene.

It has been suggested that high lens expression of the aA- and aB-crystallin genes was obtained after their duplication, which preceded the appearance of the vertebrate eye (19). It follows that the specialization for high expression in the lens was acquired independently by the aA and a B genes. The idea that the a A and a B genes arose before being selected as lens crystallins and that they independently enhanced their lens expression is consistent with their different patterns of expression (Figure 2) and, especially, with their markedly different regulatory sequences (discussed later). The mechanisms leading to specialization for lens expression are not known, but probably involved inductive responses to environmental stress.

C. NONLENS EXPRESSION OF THE (u-CRYSTALLINS

I . aB-Crystallin

The first indications of a-cry stallins' presence outside of the lens were immunological data showing cross-reactivity in the embryonic retina (43, 44). Later experiments using western immunoblotting ( 4 3 , immunocytochemistry (46), radioimmunoassay (47. 48), and Northern blot hybridization (49) established that aB-crystallin is constitutively synthesized and accumulates in many nonlens tissues under normal situations, although to a lesser extent than in the lens (see Figure 2).

The accumulation of aB-crystallin outside of the lens has been correlated with cells of high oxidative activity (46). This is consistent with the heart (which is composed of type I fibers, described below) having the highest nonlens concentration of aB-crystallin (45, 49). and with aB-crystallin concentrating in the columnar cells of the bronchial epithelium surrounding the air passageways of the lung (50). Expression of aB-crystallin in skeletal muscle is especially interesting with respect to its relationship to oxidative stress. Immuno- cytochemical localization has indicated that aB-crystallin is preferentially expressed in slow-twitch (type I ) and fast-twitch oxidative-


glycolytic (type 2A) fibers, but not in fast-twitch glycolytic (type 2B) fibers of skeletal muscle (46). The concentration of aB-crystallin decreases during disuse of the rat soleus muscle (slow-twitch) upon suspension (unless passively stretched when suspended) and in- creases in both slow and fast muscle upon passive stretching (51, 52, 53). aB-crystallin appears to play a role in stabilizing cellular structure since it is bound to the cytoskeletal components of heart (54) and skeletal muscle (53). One report has recently indicated that aB-crystallin may inhibit the assembly of intermediate filament proteins, suggesting that it may have an active role in cellular morphogenesis (55). The possibility that aB-crystallin is involved with the stabilization or organization of cellular structure is also suggested by its coappearance with ubiquitin-protein conjugates in the notochord, lens, and myotome at the time of their extensive morphological reorganization in the developing chicken embryo (56).

The kidney is another interesting location for aB-crystallin, where it has been found. in the pars recta of proximal convoluted tubules, the loops of Henle. and the inner medullary collecting ducts, and where it again has been correlated with the presence of oxidative enzymes (46). A developmental study of the rat kidney provided further support for the idea that aB-cry stallin is somehow involved with architectural reorganization by correlating its accumulation with the elongation of Henle’s loop during the first 10 days after birth (57). This links aB-crystallin with the appearance of the medullary osmotic gradient in the kidney and is consistent with the ability of aB-crystallin to be induced by osmotic stress (58).

2. aA-Crystallin

A study on the blind mole rat, which has a highly degenerate eye with a rudimentary lens buried beneath the skin, showed greater sequence conservation of the aA-crystallin gene than would be expected if aA-crystallin were entirely freed from selective constraints, suggesting a nonlens role for this protein (59). Recent immunological (47,60) and polymerase chain-reaction (PCR) (60) experiments con- ducted on the rat have shown that aA-crystallin, like aB-crystallin, is also present in numerous nonlens tissues. aA-crystallin is expressed most highly in the spleen and thymus, albeit at levels well below that observed in the lens. The appearance of both aA- and aB-crys-

162 CHRISTINA M . S A X A N D JOKAM PIATIGORSKY

tallin in the rat spleen is developmentally controlled, with aA reaching its highest concentration approximately 18 weeks after birth and a B reaching a plateau at approximately 12 weeks postpartum (47). Interestingly, the spleen is the only tissue apart from the lens in which there is more aA- than aB-crystallin. aA and aAin' mRNA have also been detected recently by PCRs in several nonlens tissues of the mouse (61).

D. EXPERIMENTAL INDUCTION OF aB-CRYSTALLIN

In addition to its constitutive expression in the lens and nonlens tissues, aB-crystallin can be experimentally induced under a variety of conditions (Table 2). aB-crystallin has been shown to accumulate transiently in NIH 3T3 cells that are expressing the Ha-ras and v- mos oncogenes (62, 63). The glucocorticoid hormone, dexamethasone, also induces aB-crystallin in NIH 3T3 cells (64). Unexpect- edly, expression of Ha-ras or v-mos for one to two weeks in the NIH 3T3 cells prevents the dexamethasone-mediated induction of aB-crystallin by an unknown mechanism (64). aB-crystallin is induced by heat shock or treatment with cadmium or sodium arsenite in NIH 3T3 cells (65) and by osmotic shock in cultured kidney cells ( 5 8 ) , consistent with aB-crystallin being a stress protein and a member of the sHSP family. The accumulation of aB-crystallin as a result of heat shock or dexamethasone treatment confers thermostability to the NIH 3T3 cells (64). HSP27 also induces thermostability in cultured mammalian cells (66). Thermostability of Chinese hamster cells appears to require the phosphorylation of HSP27 (67, 68). I t is interesting to note that, although HSP25 is also induced in the mouse NIH 3T3 cells under stress, it is not required for thermostability of

TABLE 2 Experimentally Induced Overexpression of aB-Crystallin

- oncogene (Ha-rds, v-mos) expression (63) - osmotic stress ( 5 8 ) - heat shock (65. 42) - dexamethasone treatment (42) - cadmium treatment (65) - sodium arsenite treatment (65) - stretching in skeletal muscle ( 5 I )


the cells. and aB-crystallin is able to induce the thermoresistant state by itself (42). Unlike the situation with HSP27, phosphorylation of aB-crystallin does not appear to be necessary for it to confer cellular thermostability (64).

E. EXPRESSION OF aB-CRYSTALLIN IN DISEASE

Another aspect of cYB-crystallin is that it is overexpressed in numerous diseases (69). The first hint of the connection between aB- crystallin and disease was its detection by subtractive hybridization and cDNA cloning in the brains of scrapie-infected hamsters (70). Subsequently, aB-crystallin was found associated with Rosenthal fibers of astrocytes of patients with Alexander's disease (7 I ) , where it is partially phosphorylated (721, as in the lens (731, and where it interacts with glial fibrillary acidic protein (74). Ubiquitinated inclusion bodies may be immunostained with aB-crystallin in diseased tissues, such as cortical bodies in Lewy body dimentia, Rosenthal fibers in astrocytes, and Mallory bodies in the liver (74,75), reminis- cent of the correlation of aB-crystallin with ubiquitin-protein conjugates in the chicken embryo (56) described earlier. This suggests that aB-crystallin may have some connection with protein degradation or perhaps aggregation of intermediate filaments in these tissues. aB- crystallin also appears in fibroblasts of Werner's disease, a rare inherited disorder of premature aging (76). In general, the appearance of aB-crystallin is correlated with a host of degenerative diseases, especially those associated with the nervous system, as listed in Table 3.

In addition to its association with degenerative diseases, aB-crystallin has been found in growing tissues, such as astrocytic tumors of neuroectodermal origin (77) and benign harmatomas (78). This is consistent with the overexpression of aB-crystallin in transformed NIH 3T3 cells expressing oncogenes (62,63). Other sHSPs have also been associated with cellular growth and differentiation (19).

F. CC-CRYSTALLINS~SHSPS AS MOLECULAR CHAPERONES

Recently bovine a-crystallins, mouse HSP25, and human HSP27 have been experimentally demonstrated to act as molecular chaperones (79, 80, 81, 23). They can prevent heat-induced aggregation of numerous proteins and facilitate the renaturation of chemically


TABLE 3 aB-Crystallin Expression in Disease

- adrenoleukodystrophy ( 158) - alcoholic liver disease (75) - Alexander’s disease (71) - Alzheimer’s disease (75) - amyotrophic lateral sclerosis ( I 58) - Creutzfeldt-Jacob disease (159, 160) - Huntington’s disease (158) - infectious diseases (158) - Lewy body disease (161) - metachromatic leukodystrophy ( 158) - multiple sclerosis (158) - Parkinson’s disease (158) - Pick’s disease (162) - retinoblastoma tissue and cell lines (163) - tuberous sclerosis (78) - Werner’s syndrome (76) - vascular and hypoxic encephalopathies f 158) - other primary neurodegenerative disorders ( 158)

denatured proteins. This provides a reasonable explanation for the ability of aB-crystallin/sHSPs to confer thermostability to cells. It is also likely that one of the functions of aB-crystallin in degenerative diseases is to protect against protein denaturation, perhaps slowing the progress of cellular deterioration. The idea of a protective role against protein denaturation is a less satisfying explanation for the role of aB-crystallin in cellular growth, suggesting that its possible interaction with the cytoskeleton as discussed previously is more appropriate in this instance.

The chaperone ability of a-crystallin is especially interesting with respect to its abundance in the lens. In addition to its refractive role, a-crystallin probably protects itself and other lens proteins from deteriorating throughout life. This is especially important in the central regions of the lens, where the cells have lost their nuclei and cannot renew their proteins ( I , 10). Aggregation of lens proteins leads to opacification (cataract) and must be avoided in order to maintain proper vision (82, 83). Indeed, it is likely that increased incidence of cataract with age is due, at least in part, to the loss of chaperone ability of a-crystallin (84). Thus, both the refractive and


chaperone properties of a-crystallin appear to be used in the lens as these two functions fused together during evolution of this tissue.

G. a-CRYSTALLIN HAS AUTOKINASE ACTIVITY

A proportion of both a-crystallin polypeptides are phosphorylated in vivo ( 19). The possible role(s) of phosphorylation in the function of a-crystallin are not known. Recently, purified a-crystallins have been shown to possess a CAMP-independent autokinase activity (84a). Autophosphorylation results in the production of a-crystallin polypeptides which co-isoelectrically focus with those phosphorylated in vivo. I t is possible that the autokinase ability of a-crystallin plays a role in its chaperone functions or its connection with diseases involving cellular degeneration or abnormal growth. The fact that a- crystallin possesses autokinase activity places it among the enzyme- crystallins and raises the possibility that it may be involved in one or more signal transduction pathways.

111. Regulation of a-Crystallin Gene Expression

A . aA-CRYSTALLIN

I . Purtiul Sequence Conservation and Fitnctionul Divergence

A number of studies have shown that lens-preferred aA-crystallin gene expression is conferred by sequences in the proximal 5' flanking region of the gene. Truncated fragments of the 5' flanking region of the aft-crystallin gene have been shown to drive the transcription of a fused bacterial chloramphenicol acetyltransferase (CAT) reporter gene in transfected lens cells and at the proper developmental stage in the lenses of transgenic mice (Figure 3). The high degree of lens- preferred activity of the aA-crystallin promoter has been exploited for targeting the expression of toxins and oncogenes to the lens in order to dissect lens developmental processes (Table 4). The 5' flanking sequences of the aA-crystallin gene of several species display four regions of highly conserved sequences (Figure 4). Moreover, the 5 ' flanking regions of the mouse and chicken aA-crystallin genes are able to function in a lens-preferred manner in transfected heterologous lens cells (85, 86, 87) and transgenic mice (88) (Figure 3). However, the minimal sequence required for promoter activity dif-

+ I

r- ’3

7

i

+ z z

+ + I

I 66

a-CRYSTALLIN GENE EXPRESSIVN 167

TABLE 4 Use of the crA-Crystallin promoter to Target the Expression of Toxins and

Oncogenes in the Lenses of Transgenic Mice

DNA Fragment Linked Structural Gene Phenotype References

mouse SV40 T antigen - 366/ + 46 r ich A

polyomavirus T antigen diptheria toxin A dbl oncogene human papillomavirus

human papillomavirus

HIV tat

type 16 E6 oncogene

type 16 E6 oncogene

urokinase-type plasminogen activator

hamster diptheria toxin A - 3471 + 43

C, f , M, T

lens-specific promoter activity

no effect on lens development or morphology

F, L, M

C-cataract; D-lens dysmorphology; F-interference with fiber cell differentiation; L-absent or small and vacuolated lens; M-micropthalmia; T-lens tumors.

fers appreciably in the two species (Figure 3). The chicken a A promoter needed 242 bp of 5' flanking sequence for expression in micro- injected primary lens epithelial cells of mice (87) and 162 bp of 5' flanking sequence in transfected primary chicken lens epithelial cells (88, 89). By contrast, activity of the mouse a A promoter required only 1 1 1 bp of 5' flanking sequence in the transfected primary chicken lens cells (86) and 88 bp of 5 ' flanking sequence for lens- specific expression in transgenic mice (90). This species-dependent

Figure 3. Critical deletion constructs defining minimal regulatory regions of the aA- crystallin gene. Various fragments of the 5' flanking regions of the mouse and chicken uA-crystallin gene were fused to the bacterial CAT gene. These promoter-reporter gene fusions were transfected into primary embryonic chicken lens epithelial cells or used to make transgenic mice. Open boxes represent transcriptional regulatory sequences. as notated. Promoter activity of each construct is denoted as relatively high ( + ), minimal ( + / - ). inactive ( - 1, or not assayed (nd). References: mouse gene (85. 86. 90. 114, 152); chicken gene (88, 89). Additional promoter deletion studies: mouse gene ( I I I . 127. 153. 154); chicken gene (87. 139); hamster gene (155).


difference in the requirement for regulatory sequences may be related to the observed differences in the amount (91) and developmental timing of aA-crystallin expression in the lens of the mouse (92,93) and chicken (94,95). In view of the differences in sequence requirements, we will describe our present knowledge of the molecular basis of aA-crystallin gene expression in the mouse and chicken separately.

2. Mouse aA-Crystallin Gene Regulation

a. Multiple Regulatory Elements Control Gene Expression. Several putative regulatory elements or regions have been identified by functional and protein-DNA binding experiments to be important for aA-crystallin gene expression in the mouse lens. These have been named DEl , aA-CRYBPI , TATA/PE I and PE2 (Figure 5) and correspond to the areas conserved across the aA-crystallin 5' flanking regions boxed in Figure 4.

b. The aA-CRYBPI Site. The aA-CRYBPI site ( 5 ' - GGGAAATCCC-3') is located at position - 661 - 57 of the 5' flanking sequence of the mouse gene and is embedded in a large dyad of symmetry (see Figure 5 ) , a feature indicative of transcription factor binding (96,97). The aA-CRYBPI site closely resembles the consensus binding site for the group of related transcription factors PRDII- BFI (98), MBP-I (99). HIV-EPl (loo), NF-kB (101, 102), dorsal (103), c-re1 (1041, H2TFl (105, 106), KBFl (107), EBP-I (IOS), HIVEN86 (109), and AGIE-BPI ( I 10). These regulate the expression of several genes in nonlens cells, most notably those involved in the immune response.

The 5' flanking sequence in the vicinity of position - 66/ - 57 was

Figure 4. Comparison of the 5' flanking sequences of the aA-crystallin gene of different species. The 5' flanking nucleotide sequences from the mouse (86). hamster (29). mole rat (59), human (156), and chicken (3 1) are aligned for maximal sequence identity. (*) designates identity with the mouse; ( - ) designates gaps introduced to maximize identity between sequences. The transcription initiation site for all genes is denoted by + I . Positions in the mouse sequence are denoted by - 174, - I I I , -88. -60, -34, +46. and +69, which correspond to the deletion fragments shown in Figure 3. Four regions exhibiting a high degree of sequence conservation are boxed (1, 11, Ill, IV), and the nomenclature for these sites in the mouse and chicken genes is noted below the boxes.

170

Conserved Sequences

Protein Binding

Functional Regions

Putative Regulatory Elements

Possible Transcription Factors

CHRISTINA M . SAX AND JORAM PIATIGORSKY

-111 4 4 4 - 3 4 -18 t27 +*I

+1 +*I

-151 -111 -47 -75 56 35 -12 +24 +U

-151 +1 I

-111 4 Bp 4 -31 -13 4 6 t31

-111 87 -75 -19 +24 +43

CREWATF aA-CRYBPl TFIID API, GR family NF-kB family

Figure 5 . Mouse aA-crystallin control regions. (-1 denotes sequences conserved with other species (see Figure 4); (m) denotes sequences protected by footprinting

) denotes sequences protected by footprinting that also form gel shift complexes ( I 1 I , 118, 119, 128); (0) denotes functional regions identified by rnutagenesis and expression studies (85 , 86, 1 I I , 112, 113, 114. 127, 152; Sax, unpublished); (m) denotes deduced regulatory elements. Arrows denote dyads of symmetry at the aA-CRYBPI site.

first implicated as an essential regulatory element for lens expression by being one (the proximal) of two (proximal [ - 88 to - 601 and distal [ - 1 1 1 to - 881) interacting regions required for activity of the mouse aA-crystallin promoter in transfected chicken lens cells (86). Site- directed mutagenesis of the aA-CRYBPI site eliminated the activity of the mouse aA-crystallin promoter when the mutated - 1 I1/+46 fragment was fused to the bacterial CAT reporter gene and transfected into mouse, rabbit, and chicken lens cells ( 1 1 I , 112). Cloned synthetic oligodeoxynucleotides of the aA-CRYBPI site inserted upstream of the Herpes simplex viral (HSV) thymidine kinase (tk) promoter and fused to the CAT gene activated the tk promoter in an orientation-independent manner in transfected mouse lens cells


but not in mouse fibroblasts ( I 13). Moreover, the mouse aA-crystallin - 88/ + 46 promoter fragment directed lens-specific expression in transgenic mice, while the truncated - 60/ + 46 promoter, which lacked half of the aA-CRYBPI site, did not function in the lenses of transgenic mice (1 14).

A cDNA encoding part of the aA-CRYBPI protein has been cloned from the aTN6 mouse lens cell line ( I I I ) . The aTN6 lens cells were derived from transgenic mice whose lenses were transformed with the SV40 T-antigen ( I 15; see I16 for discussion of lens cell lines in general). The expressed protein of the cloned aA- CRYBPI cDNA binds to the aA-CRYBPI site ( I 1 I ) and is the mouse homologue of the human transcription factors PRDII-BF 1 (98)/MBP- I (99). The partial sequence of aA-CRYBPI deduced from cDNA ( I I I ) and genomic ( I 17) clones shows that it contains two N-terminal and two C-terminal zinc-fingers of the C2-H2 type and a variant zinc- finger motif of the C2-H type in between them. The C-terminal zinc- fingers are followed by an acidic amino acid region. The human homologues (98, 99) of aA-CRYBPI have a similar structure. The mouse aA-CRYBPI gene appears to be at least 50 kb in length and composed of multiple exons ( I 17). Northern blots have established that the aA-CRYBPI mRNA is approximately 10 kb in length and is expressed in many different tissues, including the spleen and thymus ( I I I)-the two tissues where aA-crystallin expression is the highest aside from the lens (47, 60).

Protein-DNA binding studies using aTN4-I lens cells and L929 fibroblasts have provided further evidence that aA-CRY BPI may be used for expression of the aA-crystallin gene. First, the aA- CRYBPI site was footprinted in cells or nuclear extracts of aTN4- I lens cells in vitro by DNase I and in vivo by micrococcal nuclease and dimethylsulfate protection ( 1 18) (see Figure 5). Interestingly, similar but nonidentical footprints were obtained with extracts from L929 cells, which do not appear to express the aA-crystallin gene. Although entirely different proteins may occupy the aA-CRYBP 1 site in lens cells and fibroblasts, evidence has indicated that a modified form of aA-CRYBPI associated with high levels of expression binds to the aA-crystallin gene in the lens. An antibody against a synthetic peptide represented in the C-terminal region of aA- CRY BPI recognized proteins of 200,90, and 50 kD in western immu- noblots ( I 19). Only the 200-kD protein was found in the L929 fibro-

I72 CHRISTINA M . SAX AND JORAM PlATlGORSKY

blasts, while the 90- and SO-kDa proteins were detected in both the aTN4-I and L929 cells. Western immunoblotting of UV-crosslinked protein-DNA complexes suggested that the different sizes of aA- CRYBPI bind to the aA-CRYBPI site, including the 200-kDa form found in the L929 fibroblasts but not in the aTN4-I lens cells. Taken together, these data are consistent with the idea that modification of aA-CRY BPI by post-translational processing and/or differential RNA splicing may affect expression of the aA-crystallin gene. cDNAs isolated from libraries derived from the brain and muscle of mice have provided evidence for alternative splicing of the aA- CRYBPI primary transcript ( I 17). Alternative RNA splicing has also been reported for the human PRDII-BFl transcript (120).

It remains possible that one or more other transcription factors bind to the aA-CRYBPI site. A candidate for this is the mouse homologue of NF-kB. NF-kB and aA-CRYBP1 recognize similar binding sites (99, 121). If aA-CRYBPI is indeed necessary for expression of the mouse aA-crystallin gene, it represents an example of a ubiquitous transcription factor having a central role in tissue-preferred gene expression. The use of ubiquitous proteins to regulate tissue-specific expression is an emerging theme in gene regulation (122, 123). In this regard, it is interesting to note that three or four copies of the aA-CRYBPI site inserted upstream of the HSV tk promoter-CAT fusion gene activated transcription in several nonlens cell lines, while a single copy of this site increased expression only in lens cells ( I 13). This loss of lens-preference through aA-CRYBPI site multimerization may arise through a series of protein-protein interactions and should be considered when designing experiments attempting to identify tissue-specific control elements. aA-CRYBPI site-specific proteins, when bound to a single site in the promoter, may not activate transcription in nonlens cells, but when bound to multiple sites in the promoter may act in concert to compensate for modification/ additional factor deficiencies and allow for expression in nonlens cells.

c. The DEl Site. The DEI site (5’-CTGCTGACGGTGCAG- 3’) is located at positions - I I I/-97 in the 5 ’ flanking region of the mouse aA-crystallin gene (see Figure 5). It resembles the SV40 GTlI enhancer element (124) and the binding site for the ATFKREB family of transcription factors (125, 126). Mutagenesis of the DEI site


eliminated promoter activity in transfected lens cells ( 1 1 1 , 112). Wild-type but not mutant DE I oligodeoxynucleotides activated the proximal promoter fragment of the mouse aA-crystallin gene in transfected lens cells but not in nonlens cells (86, 127, 112). Both in vitro and in vivo footprinting assays have indicated that the DE1 site is bound by nuclear proteins isolated from lens and fibroblast cells (128, 118). However, as with the aA-CRYBPI site (discussed earlier), the DEI footprints were less pronounced using nuclear extracts from L929 fibroblasts than when using extracts from aTN4-1 lens cells, possibly suggesting a more stable DNA-protein interaction in the lens cells. Initial electrophoretic mobility shift assays indicated that multiple and similar complexes are formed between the DE1 site and nuclear extracts from aTN4-1 and L929 cells. Recent studies indicate that the DEI site is a functional CAMP responsive (CRE) site (A . Cvekl, F. Kashanchi, C. M. Sax, J . N. Brady and J . Piatigorsky, submitted). CRE consensus sequences compete for the formation of DEI electrophoretic mobility shift complexes, while UV-crosslink- ing and gel shift experiments using anti-ATFI and anti-CREB anti- sera indicate that ATFlKREB heterodimers bind to the DEI site. The introduction of DEI mutations that cause the site to deviate from a consensus CRE site eliminates promoter activity in transfected lens cells. Furthermore, forskolin and g-BrcAMP, known stimulators of CRE-dependent transcription, activate transcription from the wild type mouse aA-crystallin promoter in transfected lens cells but not from a truncated promoter lacking the DE1 site.

d. Functionally Redundant Regulatory Elements. Although site-specific mutation of either the DEI site or the aA-CRYBPI site was sufficient to eliminate activity of the - 1 1 1/+46 promoter fragment fused to the CAT gene in transfection experiments ( I 1 1 , 112) (see above), these individual mutations were still active in the lenses of transgenic mice ( I 14) (Figure 6). The mutation of both sites simul- taneously was required to eliminate promoter activity in the lenses of transgenic mice. These unexpected results indicated that the aA- CRYBPI and DEI regulatory elements are functionally redundant. It is possible, of course, that the DEI and aA-CRYBPI sites control different aspects of aA-crystallin gene expression such as developmental timing or spatial distribution, which were not examined in the transgenic mouse experiments. The results of promoter deletion

I74 CHRISTINA M . SAX AND JORAM PIATIGORSKY

-111 DE1 aA-CRYBPl TATAPE1 +1 PE2 +46 - - + + I CAT

+ - 1 CAT

CAT n I 0 ' - + n I u ' - CAT

n n I OI + CAT + Figure 6. Functional redundancy of the DEI and aA-CRYBPI sites. The - I I1/+46 mouse aA-crystallin gene fragment. containing either wild-type (0) or mutated (m) sequences in the DEI and/or aA-CRYBPI site, was fused to the bacterial CAT gene. These plasmids were transfected into lens cells (primary embryonic chicken lens epithelial cells [ 1121. the aTN4-I transformed mouse lens cell line [ I 1 I ] , and the N/ N 1003A untransformed rabbit lens cell line I 1121) or used to create transgenic mice ( I 14). Promoter activity of each construct is denoted as active ( + ) or inactive ( - ).

The solid box between the DEI and aA-CRYBPI site is a control mutation (positions - 99/ - 94).

studies supported the site-specific mutagenesis studies (Figure 3) . aA-crystallin promoter-CAT gene fusions containing the aA- CRYBPI site but not the DEI site displayed lens-specific activity in transgenic mice (90), while those lacking both sites were not active in the lens (114). This discrepancy in promoter activity between transfected lens cells and transgenic mice may be due to differences in the levels or modifications of transcription factors or their acces- sory factors in lens cells in culture and in intact lenses in mice.

In general, functional redundancy is particularly interesting with respect to evolution. It would serve to maintain tissue-specific gene expression if a regulatory site mutates during the course of evolution, Under such a circumstance, one site could compensate for another mutated or deleted site, thereby preserving gene expression in the tissue (129). In the mouse aA-crystallin gene, this redundancy may involve an interaction between either the DEI or @A-CRYBPI- bound proteins and another aA-crystallin transcription factor.


e. The PE1 and PE2 Regions. PEI and PE2 (see Figure 5) were originally defined on the basis of in vitro and in vivo footprinting studies (1 18). Recent site-directed mutagenesis experiments have proved these sites to be functionally important (C. M. Sax, unpublished). The PEI region, extending from positions - 32 to + 12, also contains the TATA box ( 1 30). In addition to being a conserved sequence across the cYA-crystallin 5' flanking region in several species, PE1 also shows sequence similarity with the analogous 5' flanking region of the aB-crystallin gene (31). This similarity is noteworthy considering the divergence of the aA- and aB-crystallin 5' flanking sequences further upstream (discussed later). Moreover, the PEI regions of the mouse aA- and aB-crystallin genes form similar electrophoretic mobility shift complexes with mouse lens cell nuclear extracts and are able to cross-compete with each other for complex formation (C. M. Sax, unpublished). The PE2 region, extending from positions + 24 to + 43, contains overlapping API and glucocorticoid response element (GCRE) consensus binding sites (131). PE2 is highly conserved in sequence and position in the mouse, hamster, mole rat, and human aA-crystallin 5' flanking regions, although the analogous region in the chicken promoter diverges in its sequence (Figure 4).

The precise roles of PEI and PE2 in aA-crystallin gene expression are not known. Other genes have tissue-specific regulatory regions associated with the TATA box (132, 133, 134) or downstream of the TATA box (135, 136). These regions may play a role in stabilizing or positioning the transcription initiation complex (137). Transgenic mice containing a mouse aA-crystallin - 34/ + 46 promoter-CAT fusion transgene do not exhibit CAT expression in the lens. Thus, the PEI- and PE2-bound proteins are not sufficient to promote transcription in the lens (90). This does not, however, preclude the possibility that PE 1 - and PE2-bound proteins direct tissue-specific expression in conjunction with other regulatory elements in the aA-crystallin promoter.

3 . Chicken aA-Crystallin Gene Regulation

a. A Complex Array of Regulatory Elements Control Gene Expression. More cis-acting regulatory elements have been identified in the 5' flanking region of the aA-crystallin gene of the chicken

176 CHRISTINA M . SAX AND JORAM PIATICORSKY

than in that of the mouse. Inasmuch as these studies were performed independently in our laboratory and the laboratory of K. Yasuda, the precise sequences identified by different methods were not ex- actly the same, and different terminologies developed, particularly within the -162/-90 region of the chicken gene. At the present time, it seems premature to adopt a single terminology, although we anticipate that this will occur in the near future as the experimental interpretations clarify and a consensus is reached.

We have defined seven putative regulatory elements in the chicken aA-crystallin gene: DE3, DE2A, DE2B, DElA, DEIB, aA- CRYBPI-like, and TATA (Figure 7). Delineation of these sequences involved correlating protein-DNA binding (in vitro DNase 1 and methylation interference footprinting and electrophoretic mobility

Conserved Sequences

-111 4 4 40-34 -18

Protein Binding

-167 -123-118 -100 -93 -87 -57 32 -22

-167 +1 +10

1 Functional Regions I

- 1 9 -103

Putative Regulatory Elements

cnOmCW--T -T C A m c z o e F c I CUCOTTCCC (rrCTnCCUAGh&Al'CCCACTAA7GCC CAGTATATATAG I I 1 7 I I I I 1 1 1 I

-153 -134 -128 -118 -114 -103-102 43-78 43-33 -22

Possible

Factors Transcription aCEFl aACRYBPClike TFIID

Figure 7. Chicken aA-crystallin control regions. (=) denotes sequences conserved with other species (see Figure 4); (m) denotes sequences protected by foot- printingonly, (=) denotes sequences that form gel shift complexes only, and ( denotes sequences protected by footprinting that also form gel shift complexes (88, 138); (0) denotes functional regions identified by mutagenesis and expression studies (88, 138); (0) denotes deduced regulatory elements. Arrows denote dyads of symmetry at the DE3, DEZA, DE2B, and aA-CRYBPI-like sites.


A.

shift assays) with functional mutagenesis studies in transfected lens cells (88, 138). Yasuda and colleagues have identified an enhancer at positions - 162 to -79 that is able to activate the basal promoter of the aA-crystallin or p-actin genes in transfected lens epithelial cells (139). Three putative regulatory elements called aCEl, aCE2, and aCE3 were defined (140). The different regulatory elements in the chicken aA-crystallin enhancer defined by us and by Yasuda's laboratory are shown in Figure 8 (a and b, respectively). We will first describe the results from our laboratory, followed by a brief discussion integrating the results of Yasuda and colleagues.

b. The aA-CRYBP1-like Site. It is much less clear whether the chicken aA-CRYBPI-like sequence plays a role in aA-crystallin

-1w140

h a d -141tl18

-153 -1 40

-144 -134 -128 -118 -114 -104 -100 -93

B. -162 -1 34

aCEl I aCE3 I

-1 35 -121

Figure 8. Comparison of nomenclature for the chicken aA-crystallin regulatory regions. The 5 ' flanking sequence of the chicken aA-crystallin gene is shown, and nucleotide positions are marked relative to the + I transcription initiation site. Two dyads of symmetry thought to be important in gene expression are shown: - 1531 - 140 (88, 140, 142) and - 1411- 118 (138). (a) The DE3. DE2A. DEZB. DEIA, and DElB regulatory elements identified by Klement et al. (138). (b) The aCEI , aCE2, and aCE3 regulatory elements identified by Matsuo et al. (139) and Matsuo and Ya- suda (140).

I78 CHRISTINA M. SAX AND JORAM PIATIGORSKY

gene expression as it appears to do in the mouse. Table 5 compares the properties of this sequence in chicken and mouse with respect to its possible use as a cis-acting regulatory site. It is possible that the chicken aA-CRYBPI-like protein regulates other genes expressed in the chicken lens. The presence of an aA-CRYBPI-like protein in the chicken lens may account for the ability of the mouse aA-crystallin promoter to function in transfected chicken lens cells (85 , 86, 112).

The chicken aA-CRYBPI-like site forms a less perfect dyad of symmetry than does the mouse aA-CRYBPI site (see Figures 5 and 8) and differs from the mouse site by a G to A residue change at position two (see Table 5). Interestingly, the consensus binding sites for the NF-kB, PRDII-BFI , and aA-CRY BPI transcription factors exhibit a striking conservation of the G residue at this position (102), and single base pair changes within the analogous site in other genes change the binding of several nuclear proteins ( I2 I ) . This single base pair difference in the aA-CRYBPI binding site of the mouse and chicken and/or the degree of imperfection of the dyad nucleotide sequence itself may result in the binding of different proteins to this site in mouse and chicken lenses. It is noteworthy in this respect that the mouse and chicken aA-CRYBPI sites did not compete for binding lens nuclear proteins of chicken (see Table 5). The recruitment of upstream regulatory sequences ( - 1621- I 1 1) in the chicken aA promoter may have eliminated the selective pressure to maintain the use of the aA-CRYBPI binding site for expression of the chicken aA-crystallin gene. It will be interesting to investigate the regulation of the mole rat and human aA-crystallin genes, which have G k

TABLE 5 Comoarison of the Mouse and Chicken aA-CRYBPI Sites

Property Mouse Chicken

sequence GGGAAATCCC (86) GAGAAATCCC (3 1) activates the tk promoter yes (113) no (113) inactivated by mutation yes ( 1 1 1 , 112) no (138, 140) footprinted by DNase I yes ( I 18) yes (138) uA-CRYBPI mRNA in lens yes ( 1 12) EMSA: competition with chicken not done yes ( 138)

EMSA: competition with mouse yes ( I 18) no (138)

yes (1 1 I )

site

site


GAAATCCC sequences in their 5’ flanking region like the chicken, and the hamster aA-crystallin gene, which has a GGGAAATCCC sequence, like the mouse.

c. The DElA/DElB Region. The chicken DEIA/DEIB region ( - I14/-91) can be compared with the DEI region ( - I1 I/- 97) in the 5’ flanking region of the mouse aA-crystallin gene (see Figure 7). DEIA( - I14/- 103)and DElB ( - 102/-91) sequences have been distinguished, in that mutation of the former in transfection experiments reduced functional activity, while mutation of the latter did not (138). Indeed, we noted that mutation of DElB actually increased promoter activity somewhat (I 38): however this was not observed by Matsuo and Yasuda (140), and additional studies are necessary in order to determine whether this has any functional significance. Moreover, in vitro DNase 1 footprinting did not reveal the binding of lens nuclear proteins to the DE I A sequence, but did reveal binding to the DElB sequence. Electrophoretic mobility shift assays did, however. indicate the specific binding of nuclear proteins to DE 1 A (138).

Although the mouse DEI and chicken DEIA/DElB elements are similar in sequence, they appear to bind different lens proteins. Com- petition experiments demonstrating that the mouse DEI site does not compete for the binding of lens nuclear proteins to the chicken DElA or DElB sites in electrophoretic mobility shift assays support the idea that the mouse and chicken elements bind different transcription factors (138). While both mouse DEI and chicken DEIA/ DEl B have the same core sequence (5’-CTGCTGAC-3’). they differ in their adjacent sequences. The importance of flanking sequences in transcription factor binding has been described (97, 141), and the flanking sequence differences between the mouse DEI and chicken DElA elements may contribute to the use of different transcription factors in the two species. A potentially important difference between DEI and DEIA/DEIB is that the mouse DEI core sequence overlaps a consensus sequence of the ATFKREB family of transcription factors (see Figure 5) (l2S, 126), while the chicken DEIA/ DElB sequence does not. In fact, recent evidence suggests that the mouse DEI site binds ATFI/CREB heterodimers (A. Cvekl, F. Ka- shanchi, C. M. Sax, J. N. Brady and J. Piatigorsky, submitted) while the chicken DElB region binds the transcription factor USF (A.

180 CHRISTINA M. SAX AND JORAM PIATICORSKY

Cvekl, C. M. Sax, J . F. Klement, E. Bresnik and J . Piatigorsky, in preparation). Thus, the mouse DEI and chicken DEIA/DEIB regions may be examples of evolutionarily conserved regulatory sites binding different transcription factors in different species. Perhaps the binding of different proteins to the mouse DEI and chicken DEI AIDE I B sites necessitates their differing interactions with additional regulatory sites-in other words, the downstream aA- CRYBPl and PE2 sites in the mouse vs. the upstream DE2A and DE2B sites (discussed next) in the chicken.

d. The DE2A and DE2B Elements. Initial deletion mutagenesis and footprinting experiments indicated that the sequences contained within the - 1531- 140 dyad of symmetry (in the DE3 region-see Figure 8) were required for regulation of the aA-crystallin gene (88, 139, 140). This was supported by the identification of a 61-kDa nuclear protein, called aCEF 1, that binds specifically to this region and is greatly enriched in the lens (139). However, further experiments showed a poor correlation between the functional and protein-binding studies of the - 153/- 140 sequence; mutation of the upstream half of the dyad did not reduce enhancer activity but had a detrimen- tal effect on protein binding, while mutation of the downstream half of the dyad reduced enhancer activity but had little effect on protein binding. These apparent inconsistencies bring into question the ac- tual role of the - 1 % - 140 dyad (138). Another interesting observa- tion by Matsuo and Yasuda (140) is that mutagenesis of the upstream half of the - 153/- 140 dyad elevated enhancer activity, while mutagenesis of the downstream half reduced enhancer activity in transfection experiments. We found that mutagenesis of either half of the dyad reduced enhancer activity (138). Clearly, further experiments are necessary in order to establish the role(s) of the - 153/- 140 dyad.

In view of this complexity, attention was directed toward a different dyad of symmetry located downstream at positions - 144 to - 118 (see Figure 8). The upstream half of this dyad ( - 1441- 134) was called DE2A, and the downstream half ( - 1281- 118) was called DE2B. It is important to note that DE2A overlaps with the downstream half of the - 1531 - 140 dyad. DE2A and DE2B formed similar complexes and cross-competed for complex formation with lens nuclear extracts, consistent with the possibility that they bind similar

a-CRYSTALLIN G E N E EXPRESSION 181

factors. Mutagenesis of DE2A and DE2B reduced both enhancer activity and protein binding, strengthening the possibility that these are important functional elements.

The aCE 1 (positions - 162/- 134). aCE2 (positions - Il9/-99). and aCE3 (positions - 13Y- 121) regions were defined by Matsuo and Yasuda (140) and are shown in Figure 8. The aCEl , aCE2, and aCE3 regions overlap with our regulatory regions described above, and mutagenesis experiments at the two laboratories have given generally similar transfection results. Matsuo and Yasuda (140). however, have tested their sequences for the ability to activate the p-actin promoter fused to the CAT reporter gene. In brief, the interaction of trimers of aCEl and aCE2 placed upstream of the p-actin promoter generated high, lens-preferred enhancer activity in transfected cells. When two sets of aCEl and aCE2 were inserted downstream of the CAT gene, enhancer activity depended upon having at least one copy of either element close to the TATA box. Individual trimers of aCEI, aCE2, or aCE3 by themselves showed little enhancer activity, consistent with the need for cooperative interaction of these regulatory elements. Trimers of aCEI combined with trimers of aCE3 gave some lens-preferred enhancer activity, but much less than that with trimers of aCEI combined with trimers of aCE2. We have speculated that the relatively poor enhancer activity of aCE3 may be due to the fact that it contains only truncated portions of dyads in DE2A and DE2B. possibly limiting its ability to bind the appropriate transcription factor(s) (138).

Although trimerization of sites is an artificial situation. it does point out that each element requires the presence of additional regulatory elements to direct lens-preferred expression, and a combina- tion of aCEl and aCE2 is sufficient to confer lens-preferred activity. The inability of aCEI, aCE2, or aCE3 to exhibit enhancer activity without multimerization suggests that, in order to direct lens-preferred promoter activity in the native gene, the single copies of aCEl and aCE2 require additional regulatory sequences, such as perhaps the aCE3 region (140). Resolution of the regulatory roles of the different sequences within the 5' flanking region of the chicken aA- crystallin gene will be greatly helped by characterization of the transcription factors that bind to these sequences.

e. The aCE1, aCE2, and aCE3 Regions.

182 CHRISTINA M. SAX AND JORAM PlATlGORSKY

Finally, a survey of crystallin genes has raised the possibility that consensus motifs for aCE I (5'-CT/AGG/CNNCCCACCAG-3') and orCE2 (5'-TGCTGACC-3') are present in a number of crystallin genes and may contribute to their high expression in the lens (140, 142). The ability of these sequences to form either unique or very pronounced complexes with lens nuclear proteins has supported this idea. Fur- ther experiments defining the cis-acting regulatory elements and their binding proteins will establish the general significance of the sequences described here.

B . aB-CRYSTALLIN

I . Comparisons qf 5' Flanking Sequences

The expression pattern of the aB-crystallin gene is much more complex than that of the aA-crystallin gene. Moreover, the abilities of aB-crystallin to protect cells against physiological stress and of its gene to be induced by numerous environmental insults also indicate that stress-responsive as well as multiple, tissue-preferred constitutive control elements are present in this crystallin/sHSP gene. An early comparison of the 5' flanking sequences showed a partial similarity between positions - 100 and - 10 of the 5' flanking sequences of the hamster cYB-crystallin gene with comparable positions of the chicken, mouse, and hamster aA-crystallin genes, and little similarity between the a B and a A genes further upstream (31). A particularly interesting sequence similarity between the a B and aA- crystallin genes of a number of species includes the TATA box and adjacent 3' sequences (PEI of the mouse aA-crystallin gene). The analogous 5' flanking sequences of the mouse sHSP25 (39, 40) and human sHSP27 (41) genes are also similar. This is noteworthy inasmuch as these mammalian sHSP genes are ancestrally related to the a-crystallins. Moreover, the mouse sHSP25 gene has been shown to be expressed more highly in the lens than in any other tissue (42) (see Figure 2), strengthening the possibility that PEI is an important regulatory element for high expression in the lens.

A recent comparison of the 5' flanking regions of the human, mouse, and rat aB-crystallin genes showed a great deal of overall similarity within the 400 nucleotides immediately upstream of the lens transcription initiation site (50). Several potential cis-regulatory


sequences of special interest that we will discuss further include the consensus heat-shock element inverted NGAAN repeats (143) and consensus AP2-like elements (CCCCAGGC) (97, 144) present in the rodent and human 5' flanking regions (33).

2. Transgenic Mice

Experiments with transgenic mice have been useful for delineating regions containing transcriptional control regions of the mouse aB- crystallin gene. Initially, an aB-crystallin minigene containing 666 bp of 5' and approximately 2,400 bp of 3' flanking sequences and lacking exon 2, portions of exons 1 and 3, and both introns was used as a transgene in transgenic mice (49). The pattern of expression of the aB-crystallin minitransgene was generally similar to that of the natural a B gene (expression in lens, skeletal muscle, heart, kidney, brain, spleen, lung). Subsequent experiments were performed with a transgene comprising a -661/+44 fragment of the aB-crystallin gene fused to the bacterial CAT gene (145). A single insert of this CAT transgene was expressed strongly in the lens and weakly in the skeletal muscle of the transgenic mouse. The a B promoter-CAT transgene was expressed highly in the lens, heart, and skeletal muscle, slightly in spleen and lung, and not at all in kidney and brain when multiple (six) copies were present in the transgenic mice. To- gether, these experiments demonstrated clearly that regulatory elements for lens and skeletal muscle are present in the 5' flanking sequence of the aB-crystallin gene and suggested that additional regulatory elements for expression in other tissues may also exist in the 3' flanking sequence of the gene. Transfection experiments using a - 537/ + 21 fragment of the human aB-crystallin gene fused to the CAT gene showed little or no expression in astrocytoma cell lines containing aB-crystallin mRNA, further suggesting that 3' regulatory elements may exist for expression of the natural aB-crystallin gene (33). Current transgenic mouse experiments using truncated fragments of the aB-crystallin 5' flanking sequence placed upstream of the CAT gene have now established that sequences between positions -426 and - 164 are essential for expression in skeletal muscle and heart, and sequences downstream of -164 are sufficient for expression in lens (145a).

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3. The aB-Crystallin Enhancer

The presence of an enhancer especially important for expression in skeletal muscle cells was revealed between positions -427 and -259 (see Figure 9) by transfection experiments using a series of fusion genes containing truncated fragments of the 5' flanking region of the mouse aB-crystallin gene, the HSV tk promoter, and the human growth hormone gene (145). The - 4271 - 259 enhancer func- tioned in either orientation and in different positions within the construct, and it showed similar low activity in the lens cells or undiffer- entiated myoblasts. However, enhancer activity increased 20- to 30- fold after the trdnsfected C2C 12 myoblasts formed differentiated rny- otubes, consistent with the large increase in aB-crystallin mRNA that occurs after differentiation of the myoblasts into myotubes (145). Deletion experiments suggested that activity of the enhancer depends on the interaction of distal ( - 426/ - 339) and proximal (-314/-257) elements.

Four functional elements (aBE-1, aBE-2, aBE-3, and MRF) were discovered in the enhancer by DNase I footprinting, electrophoretic mobility shift, and site-specific mutagenesis experiments ( 146) (see Figure 9). aBE-I, aBE-2, and aBE-3 do not contain known consensus sequences for transcription factor binding, except for an AP2- like binding sequence in aBE-2. It is not known if AP2 is used as a transcription factor by the aB-crystallin enhancer. By contrast, the MRF element (for muscle regulatory factor binding site) contains an E-box that binds MyoD, myogenin, and other members of this family of muscle-specific transcription factors (see Figure 9). Indeed. MyoD and myogenin were able to activate the enhancer fragment in NIH 3T3 cells in cotransfection experiments, although to a lesser extent than its activity in the C2C12 myotubes. This indicates that the MRF element interacts with other elements (probably aBE- I , aBE-2, and aBE-3) in the C2C12 cells to achieve full activity. It is not known

Figure 9. Mouse aB-crystallin control regions. Footprints: (0) denotes sequences footprinted by nuclear extracts from aTN4-1 lens cells, C2C12 muscle cells, and L929 fibroblasts, while (m) denotes sequences footprinted only by extracts from C2C12 muscle cells, and (0) denotes sequences footprinted only by extracts from aTN4-I lens cells (146). Putative regulatory elements: deduced by footprinting and expression studies (145. 146). The lens-specific region has been studied recently (145al. Possible transcription factors: the underlined bases represent possible inverted NGAAN re- peat sequences known to be important for HSF binding.

+

I86 CHRISTINA M . SAX AND JORAM PIATIGORSKY

which member of the MyoD/myogenin family of transcription factors is actually used by the aB-crystallin enhancer for expression of the natural gene in skeletal muscle. Recent experiments implicate myogenin over MyoD, since the former is preferentially expressed in slow oxidative muscle fibers that accumulate aB-crystallin, while the latter is preferentially expressed in fast glycolytic fibers that have little aB-crystallin ( 147) (see previous discussion of aB-crystallin expression in muscle).

aBE-I, aBE-2, and aBE-3 appear to be used as control elements for expression in both transfected lens and C2C12 muscle cells, while the MRF element appears to be muscle-specific. The aBE-I , @BE- 2, and aBE-3 elements were all protected from DNase 1 digestion by nuclear proteins of lens, muscle, and L929 fibroblast cell lines, while the MRF element was protected only by nuclear proteins of the muscle C2C12 cell line (146). Although the aBE-I , aBE-2, and aBE-3 elements placed upstream of the tk promoter/human growth hormone gene construct worked relatively inefficiently in the transfected lens cells, they might interact with downstream elements in the natural gene to become a strong enhancer in the lens as well. The proteins that occupy the regulatory elements of the a B enhancer in the lens, muscle, or fibroblast cell lines await cloning. Since L929 fibroblasts express the aB-crystallin gene at low levels, if at all (146, 148), it is possible that the binding proteins are modified or even different than those in the highly expressing lens and muscle cells. A similar situation exists for the DEI and aA-CRYBPI regulatory elements of the aA-crystallin gene, which are also occupied by nuclear proteins of fibroblasts that do not express this gene ( I 18).

4. Lens Expression

Although the aB-crystallin gene is expressed constitutively in many different tissues, it is most highly expressed in the lens where aB-crystallin plays a role in refraction. Current footprinting and transgenic mouse experiments have indicated that sequences between - 164 and +44 contain regulatory elements responsible for lens-specificity (l45a, 146). Transfection experiments using truncated fragments of the 5' flanking sequence of the mouse aB-crystallin gene fused to the CAT gene implicated the sequence between positions - 115 and +44 for promoter function in lens cells (145).


The possible involvement of the sequences downstream of the TATA box for lens expression is consistent with the conservation of these sequences in the a-crystallin and sHSP genes expressed highly in the lens. It is also noteworthy that the - 124/ - 1 1 1 sequence is similar to the aCEl region of the chicken aA-crystallin gene.

5 . Alternative Transcription Initiation Sites

Northern blot experiments on duck (149), mouse (49). and rat (71, 150) showed that there are at least two sizes of aB-crystallin mRNA that differ by 300 to 500 bases in length. In general, t he smaller mRNA predominates in the lens and most other tissues containing relatively high concentrations of aB-crystallin mRNA (i.e., heart. skeletal muscle, and kidney), while the larger mRNA predominates in tissues that have low concentrations of aB-crystallin mRNA (i.e., lung, brain, and spleen). The mature duck is an exception, where both aB-crystallin mRNAs have been observed in the lens (24, 149) and only the smaller mRNA was found in all other tissues examined, including lung (24). Low levels of the longer aB-crystallin mRNA were detected in essentially all tissues of the rat examined by North- ern blots, except for lens (150).

Analysis of aB-crystallin cDNAs from the rat brain indicated that the additional length of the longer mRNA was due to an extended 5' untranslated leader sequence (150) (see Figure 9). RNase protection experiments supported this conclusion and suggested that the upstream transcription initiation site is near position -280. We have performed primer extension, S I nuclease, and PCR transcription experiments using lung and brain RNA from the mouse and found that the results are consistent with an upstream transcription initiation site at position -474 (50). It is possible that a high degree of secondary structure within the 5' sequences of the larger aB-crystallin mRNA has complicated the analysis of this region. In vitro transcription of the cloned mouse aB-crystallin gene fragment in a HeLa cell-free extract also supported the evidence that transcription is initiated at positions + I and -474 (P. Frederikse, R. A. Dubin, J. Haynes and J . Piatigorsky, submitted). This differs from the results with the aA-crystallin gene, which showed transcription initiat- ing only from its + 1 position in a HeLa cell-free extract (128). A


minor transcription initiation site was reported by primer extension at position -49 of the aB-crystallin gene in NIH 3T3 cells transformed with v-mos and treated with dexamethasone (63). The putative transcription initiation sites at positions + I and -49 are each preceded by a putative TATA box approximately 25 bp upstream, while transcription initiation at position -474 has a poor match for a TATA box 36 bp upstream (see Figure 9).

The sequences between positions -474 and + 1 of the aB-crystallin gene are contained within the longer lunglbrain aB mRNA. Since the aB-crystallin protein is similar in size in the lung and lens ( 4 9 , which have predominantly the larger and smaller mRNA, respectively, it follows that the same AUG codon is used to initiate translation of the two mRNAs. The longer aB-crystallin mRNA has multiple out-of-frame AUG codons followed by short open reading frames in its 5' untranslated sequence. In vitro translation experiments have suggested that this 5' leader sequence acts to suppress translation (150).

6. Stress lndirction

In addition to its constitutive expression, the aB-crystallin/sHSP gene is also induced by numerous physiological stresses (see Table 2). Putative heat-shock elements (inverted NGAAN repeats) (143) are present in the 5' flanking sequence of the mouse, rat, and human aB-crystallin genes in similar relative positions (33,49, 150). In addition, there is a sequence resembling a consensus heat-shock element within the enhancer between positions - 388 and - 367 of the mouse aB-crystallin gene (see Figure 9). Binding and immunological data have supported the idea that these canonical heat-shock response elements contribute to the heat induction of the aB-crystallin gene (151). Further studies are necessary in order to identify the molecular basis of the heat-shock response of the aB-crystallin gene and to determine the sequences responsible for the induction of the aB- crystallin gene by chemical (65). oncoprotein (62, 63), or osmotic (58) stresses.

IV. Conclusions

The two a-crystallin genes are descendants of a primordial sHSP family. Both are specialized for high expression in the lens, where


their encoded proteins contribute to the refractive properties of this transparent tissue. Only the a B gene has been shown to still be inducible by heat and other physiological stresses. The expression patterns of the aA- and aB-crystallin genes are different. Although the cxA-crystallin gene is not expressed solely in the lens, it is more specialized for lens expression than is the a B gene, which is constitutively expressed in many tissues. Moreover, the aB-crystallin gene is overexpressed in many disorders, especially degenerative diseases. This may be related to the chaperone function of aB-crystallin, which can protect cells and proteins from deterioration under stress, or its autokinase activity. The innovative use of the a-crystallins for multiple functions is associated with changes in gene regulation. Transfection and transgenic mouse experiments have shown that the mouse and chicken aA-crystallin genes can function with lens preference in the homologous and heterologous species. Experi- ments have revealed a number of unexpected differences in putative regulatory elements and possible transcription factors between mice and chicken. Common and species-specific features of lens-preferred expression of the cxA-crystallin gene have not been fully resolved yet, although efforts are under way in this direction. There are at least two principal transcription initiation sites in the aB-crystallin gene that are used in a tissue-preferred manner. An enhancer with high activity in muscle and apparently lower activity in lens is present between the upstream and downstream transcription initiation sites. Multiple control elements exist between the two transcription initiation sites; some are shared by several tissues (i.e., lens and muscle), and others appear to be used selectively by specific tissues (i.e., lens or muscle). The differential expression of the a-crystallin genes in numerous tissues makes it likely that there are more regulatory elements to be discovered and that the present information is just the tip of the iceberg. Moreover, other aspects of a-crystallin gene expression, such as chromatin structure, splicing, transport, stabil- ity, and translation of mRNA are also fertile ground yet to be ex- plored.

Acknowledgments

The authors wish to thank J. Brady. A. Cvekl, P. Frederikse, R. Gopal-Srivastava, J. Haynes, J . Horwitz, M. Kantorow, and S.


Tomarev for critical comments on the manuscript, and N . Ambulos and L. Willett for assistance in compiling the references.

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